WO2013100132A1 - Peptide-compound cyclization method - Google Patents

Abstract

The present invention addresses the problem of providing an effective drug-discovery technique for tough targets for which drug discovery has heretofore been difficult. The present invention pertains to the following: a novel peptide-compound cyclization method for solving the aforementioned problem; a novel peptide compound; and a library containing said method and compound.

Description

Method for cyclization of peptide compounds

The present invention relates to a novel cyclization method for peptide compounds, a novel peptide compound and a library containing them.

In recent years, drug discovery technology development that enables drug discovery to tough targets typified by protein-protein interaction inhibition, agonists, and molecular chaperones has been attracting attention in recent years using medium molecular compounds (molecular weight 500 to 2000). (Non-Patent Document 1). It has been considered that effective inhibition of tough targets, which have been difficult to find drugs other than antibodies, is possible even with compounds having a molecular weight of 500 to 2000 (Non-patent Document 2). In addition, cases that are outside the range of rule of 5 proposed by Lipinski (most of which has a molecular weight of over 500) can be blocked by oral agents and intracellular targets, mainly natural products. (Non-patent Document 3). It is possible to realize that medium molecular compounds cannot be converted into low molecules because they can access tough targets, and that they cannot be converted into antibodies due to their ability to migrate into cells (intracellular target drug discovery or oralization is possible). It is a highly valuable molecular species.

Since most of the conventional low molecular drug discovery has been carried out in the molecular weight range of less than 500, the acquisition of Hit (hit) compound from the conventional low molecular weight compound using Tough target (HTS (High Throughput Screening)) A generic term for difficult drug targets, characterized by the absence of a deep cavity (cavity) into which small molecules can bind, such as inhibition of protein-protein interaction, such as IL-6 and IL-6R binding inhibition. Others include inhibition of RNA-protein interaction, inhibition of nucleic acid-nucleic acid interaction, etc.) Most of the middle molecules that enable drug discovery are limited to natural products. Drugs derived from natural products are still analyzed to account for 30% of First In Class (FIC) compounds, and are effective techniques. However, even if all known compounds are combined, there is a diversity of about 10 ⁶ compounds, so the target from which the active compound can be obtained is limited. In addition, the membrane permeability and metabolic stability of the active compound are often difficult, and there are 10 ⁶ kinds of molecules that can penetrate the membrane that enable drug discovery in a region where neither low molecules nor antibodies can be found. It is thought that it is far below. Even when it is desired to improve the membrane permeability and metabolic stability of the natural product Hit, it is often difficult to improve by chemical modification because complex chemical synthesis is required. For this reason, many natural products have been put on the market without being chemically modified.

By utilizing in vitro display technology that has been put to practical use in bio-drug discovery, a new group of high-molecular compounds with high diversity can be created in a short period of time. There are various limitations at present in order to develop into drug discovery. This limitation is easy to understand when compared with antibody drug discovery, which has already been put into practical use. An antibody that can generate molecules for any target from a large library has a large protein scaffold, a long variable region, and a combination of three-dimensionally diverse structures to form secondary and tertiary structures. A surface can be formed. Therefore, it is possible to create a compound that binds and inhibits many extracellular proteins with a library of about 10 ¹⁰ kinds. On the other hand, cyclic peptides, which are medium molecules obtained by utilizing biotechnology, require membrane permeability that cannot be achieved by antibodies, so chain length (molecular weight) is limited. There is a limit to three-dimensional diversity because it is limited.

It is highly valuable to create technologies that can be easily synthesized and chemically modified, have membrane permeability and metabolic stability, are structurally diverse, can produce a large number of medium molecules in a short period of time, and can evaluate their efficacy. One of the candidates for such a technique is the above-mentioned Display Library (display library) technique (can synthesize and evaluate 10 ¹² compounds at a time). The compounds that can be obtained from the display library are currently limited to peptides, but peptide pharmaceuticals are valuable chemical species that are already on the market for over 40 types (Non-Patent Document 4). Cyclosporin A is a representative example of a peptide produced by an 11-residue microorganism that inhibits an orally administrable intracellular target (cyclophilin). Peptides generally have low metabolic stability and membrane permeability, but it has also been clarified that they can be improved by non-naturalization such as peptide cyclization or N-methylation (Non-patent Document 5). ).

On the other hand, since the structure of the natural peptide changes due to main chain conversion such as non-natural peptide formation, especially N-methylation, membrane permeability to the peptide and metabolic stability are maintained while maintaining its medicinal properties using the natural peptide as the lead compound. It is extremely difficult to obtain clinical candidate compounds by imparting both sexes. A successful example of cyclization of an integrin-inhibiting peptide and clinical trials as an oral agent by non-naturalization has been reported (Non-Patent Document 6), but such drug development is brought about at the end of a long research period. There are few examples that have been made. For example, oral development of high-value pharmaceutical injections such as insulin, GLP-1 (glucagon-like peptide-1), PTH (parathyroid hormone), calcitonin has not been successful.

In recent years, it has been reported that peptides containing non-natural amino acids and hydroxycarboxylic acid derivatives (Non-Patent Document 22) can be synthesized by ribosomes, so that Display® Library containing non-natural amino acids has become a reality. In particular, it has been reported one after another that peptides containing N-methyl amino acids can be synthesized in ribosomes by utilizing tRNA that binds a non-natural amino acid and a cell-free translation system such as PureSystem (registered trademark) (Non-Patent Documents 7, 8, 9, 10, 11). An example of a challenge to Display® library containing one unnatural amino acid was also reported (Non-patent Documents 12 and 13). In addition, an example has been reported in which a display of a peptide containing N-methylamino acid is produced (Non-patent Document 23).

Investigating the conditions for achieving the compatibility between membrane permeability and metabolic stability for medium molecular peptides is also in progress. Lokey et al. Performed proline or N-methylation of a cyclic peptide consisting of six amino acids, and identified factors that affect membrane permeation using PAMPA (Parallel Artificial Membrane Permeation Assay) (Non-patent Document 14). Furthermore, a peptide with 28% BA (bioavailability) has been created in rats (Non-patent Document 15). Kessler et al. Have reported a review of N-methylation of cyclic peptides consisting of 5 or 6 amino acids to find favorable conditions for membrane permeation and metabolic stability (Non-Patent Documents 16 and 17). On the other hand, in general terms, what kind of peptide is compatible with membrane permeability and metabolic stability, and because it is more diverse, it has a higher molecular weight than the number of hit creation expected to be high (more than 7 amino acids) ) As far as we know, there are no reports on the consideration of a wide range of druglikeness conditions for medium molecular peptides.

There is room for improvement in the cyclization method to obtain medium molecular hit compounds in the display library. For example, conventional cyclization with Phage Display has been limited to the case where two Cys are SS-bonded (Non-patent Document 18). Cyclic peptides produced by the cyclization method using SS bond are metabolically unstable and not only have a short half-life in the blood, but also are weakly acidic and reduced or cleaved in the cells, leading to degradation. It is difficult to do this, and there is a possibility that the SH group generated by cleavage will form a covalent bond with a protein in the body at random. For this reason, various improvements are required from the point of druglikeness as a medium molecular peptide. It was to be done. In recent years, as a technique for improving this, a method of cyclizing two Cys via mesitylene has been reported (Non-patent Document 19). This approach results in a more stable thioether-mediated cyclization, but the effect is partial and leaves room for improvement. For example, it is widely known that thioethers are susceptible to oxidative metabolism. It has been reported that it is decomposed into RSCH ₂ R ′ → RSH + R′CHO by cytochrome P450 and metabolized to sulfoxide by flavin-containing monooxygenase (Non-patent Document 20). When the former is generated, it becomes a reactive metabolite, which leads to toxicity.
On the other hand, there have been epoch-making reports that amide cyclization, which is a drug-like cyclization method, has been realized as a peptide cyclization method (Non-Patent Document 21, Non-Patent Document 25, Non-Patent Document 26, Non-Patent Document). Reference 27) all generate a structure in which the active species obtained by cleaving the main chain amide bond and the amino acid main chain amino group are chemically reacted. It cannot be applied as a method. These techniques are useful as a technique for condensation cyclization of many natural products such as cyclosporin A with a main chain carboxylic acid and a main chain amino group. On the other hand, in the Display library, since the main chain carboxylic acid end needs to be bound to mRNA, it is not possible to use a method for generating an active species by decomposing the main chain amide bond.

In mRNA display, two new cyclization methods have been proposed so far, but a display method with improved points has not yet been established. A cyclization method (Non-Patent Document 12) by cross-linking the amino group of methionine at the N-terminal and the amino group of lysine arranged downstream (C-terminal side) with disuccinimidyl glutarate (DSG) A cyclization method (Non-patent Document 11, Patent Document 1) by introducing an amino acid derivative having a chloroacetyl group as a translation initiation amino acid and forming a thioether by intramolecular cyclization reaction by arranging Cys downstream However, the improvement of the above points was insufficient, and development of a new cyclization method instead of these was demanded. For example, in the SS cyclization method using two cysteines, it is necessary to make two amino acids specific (cysteine), but in the cyclization method by cross-linking using DSG, there are three sites including lysine. As a result, the diversity of the structure is reduced in a peptide library having a certain number of residues.
Further, it has been reported that when the structure of the cyclization site is changed, the activity (strength of drug efficacy) of the peptide having the cyclization site is greatly reduced (Non-patent Document 24). In this example, after obtaining a peptide having a cyclization site, it has been shown that it is difficult to modify the cyclization site in order to obtain a peptide having excellent membrane permeability and metabolic stability.
There is a need for peptide libraries having cyclic sites with excellent membrane permeability and metabolic stability that can be used for the development of pharmaceuticals. However, there is room for improvement in establishing such peptide libraries in various respects.

International Publication No. 2008/117833

In order to obtain peptide clinically developed compounds with all of medicinal properties, membrane permeability, and metabolic stability, peptides with membrane permeability and metabolic stability are designed and those compound groups are displayed. Realization of Display library technology that selects peptides with medicinal effects is considered effective. Hit compounds that have membrane permeability and metabolic stability to some extent can be easily synthesized and chemically modified unlike natural products, so hit compounds are the same as conventional low molecular drug discovery within rule of 5. It is possible to carry out structural optimization while keeping the structural change of the compound small, and it can be expected that a clinically developed compound can be created relatively easily. In order to establish such drug discovery technology and method, druglikeness of medium molecular peptides that are outside the scope of rule of 5 (drug-likeness: In this specification, preferably, both membrane permeability and metabolic stability are achieved. I thought that it was essential to understand the conditions that satisfy the conditions) and to construct a display library containing unnatural amino acids consisting of molecular groups that satisfy the conditions.
On the other hand, in order to effectively use the limited scale of medium molecule peptide display (which means that there is a limitation on the peptide chain length in consideration of drug likeness), peptides with completely different structures (non-natural type) It is important to include a large amount of peptides containing amino acids. By introducing many types of amino acids with different side chain properties, it is possible to expand the variable region by reducing the number of fixed sites within the limited molecular weight, and to include peptides with different main chain structures. This is valuable because it increases the likelihood of obtaining binding peptides for a variety of targets. Effective methods from this viewpoint include, for example, displaying peptides having various cyclization sites and branched peptides.
The present invention has been made in view of such circumstances, and the present invention provides a novel cyclization method and branched peptide synthesis method for peptide compounds for the construction of peptide display libraries. It is an object to provide a drug-like display library and a drug-like peptide compound.

As a result of studies to solve the above problems, the present inventors have clarified for the first time the conditions required for a drug-like cyclic peptide. In addition, the present inventors have found a method for synthesizing a display library composed of peptide compounds having highly diverse cyclized portions that satisfy the above conditions. That is, since the possibility of obtaining a drug-like peptide having a target activity and a method for synthesizing a peptide compound library having a cyclization moiety using a novel translation method and post-translational modification is further increased, A method for synthesizing a library of peptide compounds having a linear portion has been found, and it has become possible to obtain a compound showing binding and inhibition to a target molecule, thereby completing the present invention.

That is, the present invention includes the following.
[1]
A method for producing a peptide compound having a cyclic part,
1) An amino acid residue and / or an amino acid analog residue, or an acyclic peptide compound composed of an amino acid residue and / or an amino acid analog residue and an N-terminal carboxylic acid analog is encoded with the peptide compound A process of translating and synthesizing from the nucleic acid
The acyclic peptide compound comprises an amino acid residue or amino acid analog residue having a reactive site on one of the side chains on the C-terminal side, and an amino acid residue or amino acid analog having another reactive site on the N-terminal side. A body residue or an N-terminal carboxylic acid analog;
2) Combine the reaction point of the amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog on the N-terminal side with the reaction point of the amino acid residue or amino acid analog residue in the side chain on the C-terminal side And forming an amide bond or a carbon-carbon bond.
[2]
A method for producing a peptide compound having a cyclic part,
1) An amino acid residue and / or an amino acid analog residue, or an acyclic peptide compound composed of an amino acid residue and / or an amino acid analog residue and an N-terminal carboxylic acid analog is encoded with the peptide compound A process of translating and synthesizing from the nucleic acid
The acyclic peptide compound comprises an amino acid residue or amino acid analog residue having an active ester group in the side chain, and an amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog having a reaction auxiliary group near the amine. Including a body;
2) An amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog having the reaction auxiliary group and an amino acid residue or amino acid analog residue having an active ester group in the side chain are amide-bonded to form a cyclic compound The method as described in [1] including the process of obtaining.
[3]
The method according to [2], wherein the active ester is a thioester.
[4]
The method according to [2] or [3], wherein the reaction auxiliary group is an SH group.
[5]
The method according to [3] or [4], comprising a step of removing the reaction auxiliary group after the step of obtaining the cyclic compound.
[6]
The amino acid, amino acid analog or N-terminal carboxylic acid analog having a reaction auxiliary group in the vicinity of the amine is a compound N-1 or a compound N-2 represented by the following general formula: Any of the methods;

Wherein R1 represents a hydrogen atom, S—R23 (R23 represents an optionally substituted alkyl group, aryl group or aralkyl group), or a protective group for the HS group;
R2 and R3 each independently represent a hydrogen atom or an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group or cycloalkyl group; Or R2 and R3 represent a substituent that forms a ring, or R2 or R3 represents a substituent that forms a ring with R4;
R4 represents an alkylene group which may have a substituent, an arylene group which may have a substituent or a divalent aralkyl group which may have a substituent;
R11 and R12 each independently represents a single bond, an alkylene group which may have a substituent, an arylene group which may have a substituent or a divalent aralkyl which may have a substituent. Indicates a group. ).
[7]
The method according to any one of [2] to [6], wherein the amino acid or amino acid analog having an active ester group in the side chain is compound C-1 represented by the following general formula:

(In the formula, R25 represents OH, a halogen atom, OR, or SR1 (R represents Bt, At, NSu, or Pfp, and R1 represents a hydrogen atom, an alkyl group that may have a substituent, a substituted group) An aryl group which may have a group or an aralkyl group which may have a substituent, a cycloalkyl group which may have a substituent, a heteroaryl group which may have a substituent, a substituent An alkenyl group which may have a group and an alkylene group which may have a substituent.);
R26 represents an alkylene group which may have a substituent, an arylene group which may have a substituent or a divalent aralkyl group which may have a substituent;
R2 and R3 each independently represent a hydrogen atom or an alkyl group which may have a substituent. ).
[8]
The total number of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and N-terminal carboxylic acid analogs constituting the cyclic part of the peptide compound having the cyclic part, The method according to any one of [1] to [7], which is 5 to 12.
[9]
The total number of amino acid residues and / or amino acid analog residues or amino acid residues and / or amino acid analog residues and N-terminal carboxylic acid analogs constituting the peptide compound having the cyclic portion is 9 to 13 The method according to any one of [1] to [8].
[10]
A method for producing a peptide compound having a cyclic part,
1) An amino acid residue and / or an amino acid analog residue, or an acyclic peptide compound composed of an amino acid residue and / or an amino acid analog residue and an N-terminal carboxylic acid analog is encoded with the peptide compound A process of translating and synthesizing from the nucleic acid
The acyclic peptide compound is an amino acid residue or amino acid analog residue having an active ester group in the side chain and an amino acid residue having an N-terminal main chain amino group, or an amino group in the main chain or side chain. Comprising an amino acid analog residue having an N-terminal carboxylic acid analog;
2) A step of obtaining a cyclic compound by amide bonding an N-terminal amino acid residue, an N-terminal amino acid analog residue or an N-terminal carboxylic acid analog and an amino acid residue or amino acid analog having an active ester group in the side chain. The method according to [1], comprising:
[11]
The active ester group is an alkylthioester group or an aralkylthioester group, and includes a step of converting the active ester group into a more active active ester group by adding an activator after the translational synthesis in step 1). Method.
[12]
The method according to [11], wherein the activator is arylthiol or N-hydroxysuccinimide.
[13]
In the step [12], an activator having a high reactivity with the translated thioester and an activator having a high reactivity with the amine to be cyclized are added and converted into a more active active ester group. The method described.
[14]
[13] The method according to [13], which is a step of converting to an active ester group with an arylthioester and then converting to an ester group having higher activity with an oxime and a derivative thereof.
[15]
The translational synthesis of the N-terminal site in step 1) is introduced using a translation initiation tRNA acylated with a translatable amino acid other than formylmethionine, a translatable amino acid analog or a translatable N-terminal carboxylic acid analog. The method according to any one of [1] to [14], wherein the method is performed.
[16]
In the translation synthesis of the N-terminal site in step 1), the start codon is skipped, and a translatable amino acid other than Met, a translatable amino acid analog or a translatable N-terminal carboxylic acid analog is introduced into the N-terminal The method according to any one of [10] to [14], wherein the method is performed using a method.
[17]
In any one of [10] to [14], the translational synthesis of the N-terminal site in step 1) is performed using a method of cleaving the N-terminal amino acid, amino acid analog or carboxylic acid analog with aminopeptidase. The method described.
[18]
In the translation synthesis of the N-terminal site, N-terminal formyl Met is removed by treatment with methionine aminopeptidase, and other translatable amino acids, translatable amino acid analogs or translatable N-terminal carboxylic acids at the N-terminal The method according to [17], wherein the method is performed using a method for introducing an acid analog.
[19]
Translational synthesis of the N-terminal site is translated with a translation system containing norleucine instead of Met, and treated with methionine aminopeptidase to remove the N-terminal formylnorleucine and allow other translation at the N-terminus The method according to any one of [10] to [14], wherein the method is carried out using a method for introducing an amino acid, a translatable amino acid analog or a translatable N-terminal carboxylic acid analog.
[20]
The method according to any one of [17] to [19], wherein a peptide deformylase is further used to remove the N-terminal amino acid, amino acid analog or carboxylic acid analog.
[21]
The production method according to any one of [1] to [2] 0, wherein the peptide compound having a cyclic part further has a linear part.
[22]
After the step of forming the cyclic compound of step 2), the non-cyclic peptide compound includes an α-hydroxycarboxylic acid and an amino acid or amino acid analog having an amino group which may be protected in the side chain, 3) including a step of chemically reacting an α-hydroxycarboxylic acid moiety and an amino acid or amino acid analog moiety having an amino group optionally protected by a side chain to form a branched moiety [1] to [21] The method in any one of.
[23]
A method for producing a peptide compound having a cyclic part and a linear part,
1) A non-cyclic peptide compound composed of an amino acid residue and / or an amino acid analog residue, or an amino acid residue and / or an amino acid analog residue, an N-terminal carboxylic acid analog, and an α-hydroxycarboxylic acid A step of translating and synthesizing from the nucleic acid encoding the peptide compound, wherein the acyclic peptide compound is
i) an amino acid residue (or amino acid analog residue) having a reactive site in one of the side chains on the C-terminal side, and an amino acid residue or amino acid analog residue having another reactive site on the N-terminal side Or an N-terminal carboxylic acid analog,
ii) an α-hydroxycarboxylic acid having Rf5 at the α-position between the two reaction points described in i) above (Rf5 is a hydrogen atom, an optionally substituted alkyl group, an aralkyl group, a heteroaryl group, a cycloalkyl group) An amino acid residue or an amino acid analog residue having an amino group in the side chain which may be protected in an acyclic peptide compound,
A process which is an acyclic peptide compound;
2) Combine the reaction point of the amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog on the N-terminal side with the reaction point of the amino acid residue or amino acid analog residue in the side chain on the C-terminal side Cyclization reaction step;
3) Step 4) generating the thioester group by cleaving the ester bond of α-hydroxycarboxylic acid described in ii) of Step 1) and the thioester group generated in Step 3) and the method described in ii) of Step 1) A cyclization reaction step for attaching an amino group,
Including a method.
[24]
The number of amino acid residues and / or amino acid analog residues contained between the α-hydroxycarboxylic acid described in ii) of step 1) and the amino acid residue or amino acid analog residue having an amino group in the side chain is The method according to [23], which is 7 or less.
[25]
The method according to [23] or [24], wherein the α-hydroxycarboxylic acid described in ii) of step 1) is contained in the acyclic peptide compound as Cys-Pro-α-hydroxycarboxylic acid.
[26]
From [23], the amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog having another reactive site on the N-terminal side described in i) of step 1) has a reaction auxiliary group. [25] The method according to any one of [25].
[27]
The amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog having another reactive site on the N-terminal side described in i) of step 1) does not have a reaction auxiliary group, and described in ii) The amino group of the amino acid residue or amino acid analog residue having an amino group in the side chain has a protecting group, and by adding an activator, the cyclization reaction of step 2) is carried out. After the cyclization reaction and before the cyclization reaction in step 3), the method includes the step of removing the amino group-protecting group of the amino acid residue or amino acid analog residue having the amino group described in ii) in the side chain, [23] The method according to any one of [26].
[28]
A method for producing a peptide compound having a cyclic part,
1) An amino acid residue and / or an amino acid analog residue, or an acyclic peptide compound composed of an amino acid residue and / or an amino acid analog residue and an N-terminal carboxylic acid analog is encoded with the peptide compound A process of translating and synthesizing from the nucleic acid
The acyclic peptide compound comprises an amino acid residue or amino acid analog residue having a reactive site in one of the side chains, and an amino acid, amino acid analog residue or N-terminal carboxylic acid having another reactive site at the N-terminus. Comprising an acid analog;
2) A reaction point of an N-terminal amino acid residue, an N-terminal amino acid analog residue or an N-terminal carboxylic acid analog, and a reaction point of an amino acid residue or amino acid analog having one reaction point in the side chain The method according to [1], comprising a carbon-carbon bonding step.
[29]
An amino acid residue or amino acid having a carbon-carbon double bond selected as the reaction point of the N-terminal amino acid residue, N-terminal amino acid analog residue or N-terminal carboxylic acid analog and having one reactive point in the side chain [28] The method according to [28], comprising a step of selecting an aryl halide as a reaction point of an analog residue and performing a cyclization reaction by a carbon-carbon bond reaction using a transition metal as a catalyst.
[30]
[29] The method according to [29], wherein the carbon-carbon bond reaction catalyzed by a transition metal is a Heck type chemical reaction catalyzed by Pd.
[31]
Any one of [1 to [30], having a reaction point for carrying out the cyclization reaction at a position where the total of amino acids or amino acid analogs forming the cyclic part of the peptide compound having the cyclic part is 5 to 12 The method described in 1.
[32]
[31] The method according to [31], wherein the total number of amino acids and amino acid analog residues of the peptide compound having a cyclic moiety is 9 to 13.
[33]
The peptide compound according to any one of [1] to [32], wherein a peptide compound-nucleic acid complex is produced in which the C-terminus of the peptide compound and the template used for translational synthesis are bonded via a spacer. Method.
[34]
The peptide compound-nucleic acid complex is synthesized using a nucleic acid in which puromycin is bound to the 3 ′ end of a nucleic acid encoding an acyclic peptide compound used for translational synthesis via a linker, [33] the method of.
[35]
The method according to [33] or [34], wherein the spacer is a peptide, RNA, DNA, or a polymer of hexaethylene glycol, or a combination thereof.
[36]
The method according to any one of [1] to [35], wherein the peptide compound is produced by translating a nucleic acid library comprising a plurality of nucleic acids having different sequences.
[37]
A peptide compound or a peptide compound-nucleic acid complex produced by the production method according to any one of [1] to [36].
[38]
A library comprising a plurality of peptide compounds according to [37] or peptide compounds-nucleic acid complexes having different structures.
[39]
A peptide compound having a cyclic part, which has the following (i) to (iv):
(I) the total number of amino acids and amino acid analogs is 9 to 13 including a cyclic part consisting of 5 to 12 total amino acid and amino acid analog residues;
(Ii) at least two N-substituted amino acids and at least one amino acid that is not N-substituted,
(Iii) The ClogP value is 6 or more,
(Iv) The bond of the amino acid or amino acid analog forming the cyclic part is at least a bond comprising an active ester group in the side chain of the amino acid or amino acid analog and the amine group of another amino acid or amino acid analog Have one.
[40]
Amino acids and amino acid analogs contained in peptide compounds are amino acids or amino acid analogs capable of translational synthesis, or amino acids or amino acids obtained by chemically modifying the translatable amino acids or side chains or N-substitution sites of amino acid analogs The peptide compound according to [39], which is an amino acid or an amino acid analog selected from derivatives.
[41]
Furthermore, the peptide compound according to [39] or [40], further comprising at least one linear moiety consisting of 1 to 8 in total of amino acid and amino acid analog residues.
[42]
The peptide compound according to any one of [39] to [41], wherein the amino acid or amino acid analog forming the cyclic part is an amide bond or a carbon-carbon bond.
[43]
The peptide compound according to any one of [39] to [42], wherein the cyclic part includes an intersection unit represented by the following general formula (I):

(The symbols in the formula have the following meanings;
R51 is a C1-C6 alkyl group optionally having a substituent, a C5-C10 aryl group, an aralkyl group, an ester group, or an amide represented by the formula 1,
R52 is an optionally substituted C1-C6 alkyl group, an aryl group, or an aralkyl group,
R53 is an optionally substituted C1-C6 alkyl group, or a hydrogen atom, or R53 and R51 are bonded to each other to form a C3-C5 alkylene group, which contains a nitrogen atom. It may form a 7-membered ring,
R54 is a peptide composed of 0-8 amino acid residues,
R55 is a C1-C6 alkyl group which may have a substituent, a C5-C10 aryl group, an aralkyl group, an ester group, an amide group which may have a substituent,
* Indicates a binding site in the annular portion. ).
[44]
[39] A pharmaceutical composition comprising the peptide compound according to any one of [43].
[45]
[44] The pharmaceutical composition of [44], wherein the pharmaceutical composition is an oral preparation.
[46]
[39] A method for producing a peptide compound according to any one of [45], comprising steps (i) to (v):
(I) A non-cyclic peptide compound having a total number of amino acids and amino acid analogs of 9 to 13 is synthesized by translation, and the non-cyclic peptide compound and a complex having a nucleic acid sequence encoding the non-cyclic peptide compound are bound via a linker. Forming a non-cyclic peptide compound-nucleic acid complex;
(Ii) The complex acyclic peptide compound translated and synthesized in step (i) is cyclized by an amide bond or a carbon-carbon bond, and the total number of amino acids in the cyclic portion and amino acid analogs is 5-12. Forming a cyclic compound comprising: and
(Iii) A step of contacting the peptide compound-nucleic acid complex library having a cyclic moiety obtained in step (ii) with a biological molecule and selecting a complex having binding activity to the biological molecule.
[47]
Furthermore, the manufacturing method as described in [46] including the following processes;
(Iv) obtaining sequence information of the peptide compound from the nucleic acid sequence of the complex selected in the step (iii), and
(V) A step of chemically synthesizing the peptide compound based on the sequence information obtained in the step (iv).
[48]
After the step of forming the cyclic compound of step (ii), wherein the acyclic peptide compound comprises an α-hydroxycarboxylic acid and an amino acid or amino acid analog having an amino group in the side chain which may be protected; The method according to [46] or [47], comprising a step of chemically reacting an α-hydroxycarboxylic acid moiety with an amino acid having an amino group in the side chain or an amino acid analog moiety to form a branched moiety.
[49]
The production method according to any one of [46] to [48], wherein the in vivo molecule is a molecule that does not have a region to which a compound having a molecular weight of less than 500 can bind.
[50]
The production method according to any one of [46] to [49], wherein the complex having binding activity to the biological molecule further has an activity of inhibiting the binding of the biological molecule to another biological molecule. .
[51]
The amino acid or amino acid analog on the N-terminal side that undergoes a cyclization reaction is an amino acid or amino acid analog selected from the compound represented by Compound N-1 or Compound N-2, and the amino acid or amino acid analog on the C-terminal side Is an amino acid or an amino acid analog selected from the compound represented by the compound C-1, and includes a step of removing the reaction auxiliary group after the step of obtaining the cyclic compound of step (ii). 50].
[52]
The nucleic acid sequence has a spacer at the 3 ′ end, and the C-terminus of the peptide compound to be translated and synthesized forms a complex with the nucleic acid sequence via the spacer, according to any of [46] to [51] The manufacturing method as described.
[53]
The peptide compound-nucleic acid complex is synthesized using a nucleic acid in which puromycin is bound to the 3 ′ end of a nucleic acid encoding an acyclic peptide compound used for translational synthesis via a linker, according to [52]. the method of.
[54]
[52] The production method according to [53], wherein the spacer is a peptide, RNA, DNA, or a polymer of hexaethylene glycol.

According to the present invention, a drug compound having a drug-like (particularly membrane permeability and metabolic stability) cyclic part or a peptide compound having a cyclic part and a linear part, which can be translationally synthesized, and A display library is provided. In addition, since the display library of the present invention is a library rich in diversity, there is a high probability that a hit compound for a desired target molecule can be obtained. Furthermore, since the hit compound obtained from the display library of the present invention already has excellent membrane permeability and metabolic stability, the conventional low molecular weight compound does not undergo a large structural change. It is possible to efficiently carry out optimization as a pharmaceutical product in the same way as the above idea. Thus, the present invention provides Scaffold for new drugs different from low molecular weight compounds and antibodies that have been known as drugs, and provides a new drug discovery system for efficiently creating drugs. is there.

[Correction 28.02.2013 based on Rule 91]
FIG. 2 is a diagram showing a general method for synthesizing aminoacylated pdCpA compounds in which side chain carboxylic acids are active esterified. It is the figure which showed the mass-spectrum analysis of the translation product of mRNA which codes the peptide sequence P-1 containing Glu (SBn). It is the figure which showed having analyzed the mass spectrum of the translation product of mRNA which codes the peptide sequence P-2 containing Asp (SMe). In the translation of Asp (SMe), a compound de-MeSHed from the translated full-length peptide was detected as the main product (translated peptide P-3). FIG. 6 is a diagram showing the production of peptide P-4 by hydrolysis of translated peptide P-6. It is the figure which showed translation of the peptide sequence which does not contain Tyr but contains thioester. It is the figure which showed the translational synthesis of the thioester containing peptide which does not have an N terminal amino group. FIG. 3 is a diagram showing translational synthesis of a model peptide in which an amino acid N-alkylated into a C-terminal amino acid following an amino acid whose side chain is thioesterified is introduced. It is the figure which showed the comparison of the chemical reactivity of a thioester, a glycine derivative, or a cysteine derivative. FIG. 8 is a diagram showing a continuation of FIG. 8-1. FIG. 3 shows the synthesis of amide 5d-1 by reaction of thioester 5b-1 with a glycine derivative under the imidazole addition condition. FIG. 2 shows a general method for synthesizing aminoacylated pdCpA of a cysteine derivative. FIG. 2 is a diagram showing a synthesis example of aminoacylated pdCpA of a cysteine derivative. It is the figure which showed the peptide translation synthesis which made Cys (StBu) N terminal. FIG. 3 is a graph showing the stability of compound 2n-A and compound 2e-A in a translation simulation solution. It is the figure which showed the efficient translation introduction | transduction of N terminal Cys which utilized Initiation read-through actively. It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-15: Phe). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-16: Leu). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-17: Tyr). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-18: Cys). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-19: Trp). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-20: Leu). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-21: Leu). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-22: Pro). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-23: His). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-24: Gln). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-25: Arg). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-26: Arg). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-27: Ile). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-29: Asn). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-30: Ser). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-32: Val). It is the figure which showed the mass spectrum of the translation product which encoded various amino acids in the 3rd codon immediately after Cys (P-33: Ala). It is the figure which showed the general synthetic method of the non-natural type amino acid with SH group other than cysteine, and its aminoacylated pdCpA. It is the figure which showed the mass spectrum of the translation peptide using the stabilized aminoacylated tRNA containing SH group (P-34: tBuSSEtGly, P-35: tBuSSEtβAla, P-36: tBuSSEtGABA, [M + H] is the target. Compound, S deprotection is a compound in which the protecting group attached to the SH group of the target compound has been eliminated, indicating that the target compound has been translated. Read-through was initiated from the second Thr Translation product, by-product) It is the figure which showed the mass spectrum of the translation peptide using the stabilized aminoacylated tRNA containing SH group (P-41: Cys (StBu), P-40: PenCys (StBu), P-38: NVOC-Cys) (StBu)). It is the figure which showed the mass spectrum of the translation synthesis | combination using the method of introduce | transducing amino acids other than methionine, an amino acid analog, or N terminal carboxylic acid analog into the N terminal, and an amide cyclization peptide. It is the figure which showed the mass spectrum of the translation using Initiation read-through and an amide cyclization peptide (NCL: target compound). It is the figure which showed the mass spectrum of the translation using Initiation read-through and an amide cyclization peptide (NCL: target compound). It is the figure which showed the MS and MS / MS analysis for the translation using Initiation read-through and the structure determination of an amide cyclization peptide. FIG. 32 is a diagram showing a continuation of FIG. 30-1. It is the figure which showed the radical desulfurization reaction using a model substrate. It is the figure which showed the conditions examination of the radical desulfurization reaction using a model substrate. FIG. 5 is a diagram showing desulfurization reaction conditions for translated peptide P-50 and a mass spectrum of the obtained peptide P-51. It is the figure which showed the influence evaluation to the protein of desulfurization reaction. It is the figure which showed the mass chromatogram of the metabolite. It is a figure which shows the MS / MS spectrum of Peak1. It is a figure which shows the MS / MS spectrum of Peak2. It is a figure which shows the MS / MS spectrum of Peak3. It is a figure which shows the MS / MS spectrum of Peak4. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound P-136 by cyclization reaction by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound P-136 by cyclization reaction by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound P-137 by a cyclization reaction by LCMS. It is a figure which shows the analysis result by the mass spectrum of the translation reaction material obtained by the translational synthesis of the peptide containing the benzylthioesterified aspartic acid derivative. It is a figure which shows the analysis result by the mass spectrum of the product obtained by the amide cyclization experiment of the peptide using the thioester on translation peptide P141, and N terminal alpha-amino group. 2 is a graph showing a comparison of synthesis conditions of Compound 10. FIG. It is a figure which shows the result of having analyzed the production | generation reaction of compound P-151 by LCMS. It is a figure which shows the result of the reverse transcription reaction which used the mRNA-peptide fusion molecule | numerator after cyclization reaction as a template. It is a figure which shows the analysis result by the MALDI-MS of the peptide containing N-terminal Phe, Ala and the aspartic acid derivative by which benzylthio esterification was carried out. It is a figure which shows the analysis result by the MALDI-MS of the peptide containing N-terminal Phe, Ala and the aspartic acid derivative by which benzylthio esterification was carried out. It is a figure which shows the result of having evaluated RNA stability in cyclization reaction conditions by electrophoresis. It is a figure which shows the analysis result by electrophoresis of the reaction product of a peptide cyclization reaction. Results of MALDI-MS analysis of cyclized peptides using peptides containing aspartic acid derivatives that have been translationally initiated with norleucine and containing benzylthioester aspartic acid in the side chain, and post-translational initiation amino acid removal and peptide cyclization without the use of reaction auxiliary groups FIG. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP-606 by LCMS. It is a figure which shows the analysis result by the MALDI-MS of the cyclic peptide (compound P-H1) which has a cystenyl prolyl ester sequence and a side chain amino group. It is a figure which shows the analysis result by the MALDI-MS of the product in the intramolecular branched peptide (linear part 2) production | generation reaction using translated peptide P-H1. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP-606 by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP-606 by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP-606 by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP-607 by LCMS. Mass chromatogram before the desulfurization reaction (compound SP606) (upper row) and mass chromatogram (lower row) obtained by integrating and averaging the retention time from 0.34 minutes to 0.39 minutes after 3 hours of desulfurization reaction (analysis condition SQD FA05) FIG. Accumulating within this range, the molecular weight should be observed if not only the target compound and reaction raw materials but also by-products with similar structures are present, so the overall reaction selectivity must be accurately evaluated. Is possible. This is not limited to FIG. 56, and all of the time ranges integrated by a mass spectrum in a certain region comply with this intention. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP-607 by LCMS. Mass chromatogram before the desulfurization reaction (compound SP606) (upper row) and mass chromatogram (lower row) obtained by integrating and averaging the retention time from 0.34 minutes to 0.39 minutes after 3 hours of desulfurization reaction (analysis condition SQD FA05) FIG. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP618 by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP619 by LCMS. FIG. 61 is a mass chromatograph of LCMS 0.3 to 0.6 minutes. The peptide components are all eluted from 0.3 minutes to 0.6 minutes under the present analysis conditions. Therefore, as a result of accumulating and averaging mass chromatographs from 0.3 to 0.6 minutes for the purpose of evaluating the selectivity of the reaction, it was shown that this reaction was selectively new. FIG. FIG. 62 shows peptide P-E1 (peak I) containing an N-terminal formylmethionine, thioester cyclized with a side chain thiol group and a carboxylic acid, and the N-terminal formylmethionine removed, resulting in an exposed N-terminal amino acid. FIG. 7 is a diagram showing the result of MALDI-MS analysis of compound P-E2 (peak II) amide-cyclized with a group nitrogen atom and an Asp side chain carboxylic acid. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP618 by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP619 by LCMS. FIG. 65 is a mass chromatograph of LCMS 0.3 to 0.6 minutes. The peptide components are all eluted from 0.3 minutes to 0.6 minutes under the present analysis conditions. Therefore, the mass chromatographs from 0.3 to 0.6 minutes are integrated and averaged for the purpose of evaluating the selectivity of the reaction. It is a guess figure of the CLOGP value by the virtual library using computer simulation, the number of NMe amino acids, the average value of molecular weight, and distribution. It is a guess figure of the CLOGP value by the virtual library using computer simulation, the number of NMe amino acids, the average value of molecular weight, and distribution. LCMS is a mass chromatograph averaged over the entire range. LCMS is a mass chromatograph averaged over the entire range. LCMS is a mass chromatograph averaged over the entire range. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP664 by LCMS. It is a figure which shows the result of having analyzed the production | generation reaction liquid of compound SP672 by LCMS. It is a figure which shows the inhibitory activity of the cell proliferation by hIL-6 and shIL-6R of the compound (SP854, SP855, SP857, SP859) obtained in Example 26. It is a figure which shows the analysis result by electrophoresis of the reaction product containing the peptide-RNA complex which has an intramolecular branched peptide (linear part 2). It is a figure which shows the result by the MALDI-MS analysis of a translation reaction material. It is a figure which shows the analysis result by the mass chromatogram and mass spectrum of a translation reaction material. It is a figure which shows the analysis result by the mass chromatogram and mass spectrum of a translation reaction material. It is a figure which shows the analysis result by the mass chromatogram and mass spectrum of a translation reaction material. It is a figure which shows the analysis result by the mass chromatogram and mass spectrum of a translation reaction material. It is a figure which shows the result of having evaluated RNA stability by side chain amino group deprotection conditions by electrophoresis. It is a figure which shows the result of LC / MS analysis of the sample obtained by processing the RNase-treated sample. It is a figure which shows the interaction of the protein and peptide which consist of 13 residues which formed the cyclic | annular form by ten amino acids and branched three amino acids. It is a figure which shows the interaction of the peptide and protein which consist of 13 residues which formed the cyclic | annular form by ten amino acids and branched three amino acids (left side). It is a figure which shows the interaction of the peptide and protein which form a ring with 13 amino acids (right side). It is a figure which shows the result of having evaluated RNA stability by side chain amino group deprotection conditions by electrophoresis.

<Peptide compound>
Peptide compound having a cyclic part The peptide compound having a cyclic part of the present invention is a compound formed by amide bond or ester bond of an amino acid or an amino acid analog, and the cyclic part forms an amide bond or a carbon-carbon bond. A compound cyclized through a covalent bond such as a reaction. A compound obtained by further chemically modifying the compound is also included in the peptide compound of the present invention. The peptide compound of the present invention may have a linear portion, and can be described as, for example, Scheme A (Schemes A-1 and A-2). The peptide compound having a cyclic part may further have a linear part. The number of amide bonds or ester bonds (the number and length of amino acids or amino acid analogs) is not particularly limited, but when it has a linear part, it is preferably within 30 residues including the cyclic part and the linear part. In order to obtain high membrane permeability, the total number of amino acids including the cyclization site and the linear site is more preferably 13 residues or less. In order to obtain high metabolic stability, the total number of amino acids is more preferably 9 or more. In consideration of compatibility between membrane permeability and metabolic stability (drug likeness), in addition to the above, the number of amino acids and amino acid analogs constituting the cyclic part is preferably 5-12. Further, in addition to the above description, the number of amino acids and amino acid analogs constituting the cyclic part is more preferably 5 to 11, and further preferably 7 to 11 residues. 9 to 11 residues are particularly preferred. The number of amino acids and amino acid analogs (number of units) in the linear portion is preferably 0-8. Furthermore, 0 to 3 are preferable. In the present application, unless specifically limited, amino acids may include amino acid analogs. Here, “druglikeness” or “druglike” means that a peptide compound targets at least an oral agent, or an intracellular region of an intracellular protein, nucleic acid, membrane protein or a transmembrane domain of a membrane protein. In this case, it means having membrane permeability and metabolic stability that can be used as a medicine.

The cyclic part of the peptide compound having a cyclic part of the present invention is not particularly limited as long as it is a peptide that forms a ring, but the post-translational cyclization part has a functional group capable of satisfying both membrane permeability and metabolic stability (Druglikeness). It must be the cyclization unit that forms. If it is such a cyclization method, it will not specifically limit. Examples thereof include an amide bond formed from a carboxylic acid and an amine, and a carbon-carbon bond reaction using a transition metal such as a Suzuki reaction, a Heck reaction (Heck reaction), and a Sonogashira reaction as a catalyst. Accordingly, the peptide compound of the present invention contains at least one set of functional groups capable of these coupling reactions. In particular, from the viewpoint of metabolic stability, a functional group that forms an amide bond by a binding reaction is preferably included.

As the cyclic part of the peptide compound of the present invention, for example, a cyclic part formed by cyclization by a chemical reaction after translation synthesis as shown in Scheme A is preferable. Furthermore, a circular part that can be formed under reaction conditions that do not affect nucleic acids such as RNA and DNA after translation is preferred.

The formation of the annular portion is preferably a drug-like cyclization. Drag-like cyclization means a bond in which the bond produced is drug-like. For example, it is preferable that the bond includes a heteroatom that can be easily oxidized and does not include a bond that hinders metabolic stability. Examples of the bond generated by cyclization include an amide bond formed by a bond between an active ester and an amine, and a bond generated by a Heck reaction product formed by a carbon-carbon double bond and an aryl halide. Since these require a reactive functional group in the triangular unit (cyclized N-terminal unit) or crossing unit described in Scheme A, amino acids suitable for drug-like are not necessarily selected for the triangular unit or crossing unit. However, it is converted into a compound having a drug-like functional group after post-translational modification. In the present invention, such a bond is also included in the drug-like cyclized bond.

The curve part of Scheme A is a site that is cyclized after translation (post-translational cyclization part), and this part is a variety of post-translational modification chemical reactions represented by carbon-carbon bond formation reactions such as amide bond or Heck reaction. To form an annular portion. In this specification, “translational synthesis” means that a peptide compound is translated and synthesized from a nucleic acid (eg, DNA, RNA) encoding the peptide compound. Translation is a process of obtaining a linear peptide by repeating amide bond or ester bond reaction using mRNA as a template by the action of ribosome.
Post-translational modification refers to a chemical reaction that occurs after translation automatically or by adding another reagent other than the action of ribosome, and includes, for example, a cyclization reaction and a deprotection reaction.
Post-translational cyclization involves a ring-forming reaction among post-translational modifications. (Scheme A: A scheme for explaining the peptide compound of the present invention. The white circle unit, the black circle unit, the triangular unit, and the square unit each mean an amino acid or an amino acid analog. A triangular unit may be an N-terminal carboxylic acid analog, for example, each of the eight black circle units may be a different type of amino acid or an amino acid analog, some of which Alternatively, the amino acid or amino acid analog may be chemically or skeleton-converted to a compound having another skeleton by chemical modification that can be performed after translation. 1 unit is the amino acid or amino acid analog at the end of post-translational modification. However, this also includes those in which an amino acid or amino acid analog translated by one tRNA is chemically or skeleton-converted to a compound having another skeleton by post-translational modification. In the present application, unless otherwise specified, amino acids include amino acid analogs, and in this specification, post-translational cyclization may be simply referred to as cyclization. is there.

For example, in the scheme A-1, the cyclic part is one triangular (▲) unit (residue) (cyclized N-terminal unit), eight black circle (●) units (cyclic part main chain unit) and one It is a part composed of white circle (◯) units (intersection units), and the straight chain part is a part composed of six square (■) units (straight chain main chain units). In scheme A-2, the annular part is a part composed of one triangular unit, eight black circle units and one crossing unit, and the straight chain part is a part composed of four squares and three square units.

In the present invention, the crossing unit refers to a functional group possessed by the amino acid or amino acid analog of the triangular unit in the peptide compound on the amino acid side chain in the peptide compound before cyclization (non-cyclized peptide compound) formed after translation. An amino acid or an amino acid analog having a functional group possessed by an N-terminal carboxylic acid analog and a functional group that is cyclized by a chemical reaction, particularly if it has a functional group necessary for cyclization with the triangular unit It is not limited. The white circle unit in Scheme A corresponds to this. The crossing unit is preferably selected from the above amino acids or amino acid analogs and capable of translation from a nucleic acid. Moreover, even if the translation itself is difficult, it is not essential that the derivative itself is translated when the derivative is translated. For example, when Asp (SBn) is translated, a compound in which the side chain methylene chain of Asp is freely substituted is allowed as an intersection unit (for example, R28 and R29 of compound C-3 are not translated). May be). In the crossover unit, the amino group and carboxyl group of the main chain are used for covalent bond formation in translation synthesis, and the third functional group is necessary for post-translational cyclization, so that it has a total of three or more functional groups. There is a need. In this, the posttranslational cyclization site is cyclized by the functional group of the side chain site of the crossing unit.

Here, the amino acid or amino acid analog having a functional group that cyclizes with the crossing unit, or the N-terminal carboxylic acid analog is not particularly limited as long as it has a functional group that cyclizes with the crossing unit. This corresponds to the triangular unit of Scheme A. For example, in the scheme A, the triangular unit is arranged at the N-terminal. In such an example, when an amino acid is selected as the triangular unit, the main chain amino group can be used as the cyclized functional group. For example, when an active ester is used as the crossing unit, post-translational cyclization can be performed by the main chain amino group of the triangular unit and an amide bond. Thus, when the main chain amino group is utilized as a reactive functional group, the side chain of the triangular unit may not have a reactive functional group. Reaction auxiliary groups such as SH groups (thiol groups) can also be introduced into the side chain. When an amino acid analog is used as the triangular unit, the hydroxyl group of the main chain may be used as a reactive functional group, or a reactive functional group arranged in a side chain may be used. In addition, when an N-terminal carboxylic acid analog is used, an amino group or a hydroxyl group may be used as a reactive functional group as described above, but as a free reactive unit having no amino group or hydroxyl group. Various functional groups can be introduced. The triangular unit is preferably selected from the above amino acids, amino acid analogs, or N-terminal carboxylic acid analogs and translated. In addition, even if it is difficult to translate itself, it is not essential that the derivative is translated as in the case of the intersection unit.

The crossing unit and the triangular unit can be incorporated at a desired position in the peptide compound before cyclization as long as they can be cyclized, but cyclization or post-translational modification after cyclization is applied. It is preferable that the amino acid, amino acid analog or N-terminal carboxylic acid analog in the subsequent cyclic site is incorporated at a position where the total number is 5 to 12. Furthermore, the amino acid or amino acid analog or N-terminal carboxylic acid analog of the cyclic part after cyclization or post-translational modification after cyclization is incorporated at a position where the total number is 5 to 11. It is preferable.

In addition, the triangular unit is arranged at the N-terminal in Scheme A, but it can be arranged at a position other than the N-terminal. In that case, the position needs to be arranged on the N-terminal side from the intersection unit. When the triangular unit is arranged at a position other than the N-terminal, the triangular unit is selected from amino acids and amino acid analogs, and has a functional group that cyclizes with the crossing unit in the side chain.

The black circle unit and the square unit are selected from amino acids or amino acid analogs. Also included are chemical structures that can be produced by post-translational modification after translation with an amino acid or amino acid analog (eg, the structure of the straight chain portion 2). When an amino acid is selected, the black circle unit is not particularly limited, but is preferably selected from a drug-like amino acid or an amino acid that has a reactive functional group and is chemically reacted by post-translational modification to be converted into a drug-like functional group. (For example, lysine is mentioned as the amino acid residue of the straight chain part 2). When selected from amino acid analogs, the black circle unit is not particularly limited, but preferably a drug-like amino acid analog or an amino acid analog having various reactive functional groups on the side chain is converted into a reactive functional group by post-translational modification. Selected from amino acid analogs that are chemically modified and converted to drug-like amino acid analogs.

The number of straight chain portions (the number of branches) is not particularly limited, and may be one as shown in Scheme A-1 or two or more as shown in Scheme A-2. Further, the compound may be a compound in which the number of square units in Scheme A-1 is 0, or may be a compound in which the number of straight chain portions 1 is 0 in the square units in Scheme A-2. By having a linear part, it is possible to strengthen the function of the peptide compound having a cyclic part of the present invention. For example, when the peptide compound of the present invention is used to inhibit the binding between a certain receptor and a ligand, the peptide compound has a linear portion. The binding activity to a receptor or a ligand can be increased. By enhancing the binding activity, it is possible to enhance the binding inhibition effect between the receptor and the ligand. In particular, the linear portion of the present invention can be provided at a desired position of the cyclic portion, for example, according to the method described later, and the linear portion is provided at the optimum position for obtaining a higher function. The obtained peptide compound can be obtained (hereinafter, linear part 2).

These linear portions are also preferable for obtaining a peptide compound having a desired activity more efficiently from a library of peptide compounds having a cyclic portion. Peptide compounds having a desired activity include those having binding activity to the target substance, those having an action of inhibiting the function of the target substance, those having an action of activating the function of the target substance, Examples include those having the function of changing the function, and the function can be selected from these according to the purpose. When the peptide compound having a cyclic part of the present invention has binding activity to a target substance, these peptide compounds are excellent in membrane permeability and lipid stability. For example, by labeling the peptide compound In addition to in vivo, it is possible to monitor the distribution of target substances in cells in real time. Further, when the target substance is a causative factor of the disease, it may be used for diagnosis of the disease. In addition, when the peptide compound having a cyclic part of the present invention has an effect of inhibiting, activating or changing the function of the target substance, for example, when the target substance is a causative factor of the disease, Available. In addition, for example, when the peptide compound having a cyclic part of the present invention has the straight chain part 2, it is possible to improve the acquisition ratio of the inhibitory compound as compared with the case where there is no linear part. In the case of a peptide compound having a 13-residue cyclic moiety that binds to protein A and inhibits the protein-protein interaction between protein A and protein B, the peptide contact site in protein A to which the peptide compound is bound is determined. This is shown on the right side of FIG. Since it is a cyclic compound, when the contact region of peptide and protein A is approximated by a circle, it can be determined that the diameter is about 3 to 5 residues. When protein B binds to this contact area, it is possible to effectively inhibit the protein-protein interaction between protein A and protein B. When protein B binds to protein A outside this contact area Therefore, there is a possibility that effective inhibition cannot be obtained (so-called allosteric inhibition examples are rarely reported as protein-protein interaction inhibition examples).

Considering this, it is important to obtain peptide compounds that bind to proteins in as wide a region as possible. On the other hand, since the total number of amino acids that can permeate through the membrane is limited, the straight chain portion is effective. For example, FIG. 82-1 shows the same 13-residue cyclic peptide as above, but a peptide in which a cyclic structure was constructed with 10 residues and the remaining 3 were branched. In particular, since the straight chain part 2 can be added to any position of the peptide compound according to the method described later, not only the four branch points in FIG. Points can be acquired, but if four are overlapped, the contact area is expanded to the area shown on the left in FIG. In spite of acting at the same place (hole), by overlapping with the binding region of protein A and protein B, the ratio of obtaining a peptide compound having a function such as an inhibitor is increased.

In the present specification, “amino acid” and “amino acid analog” constituting a peptide compound may be referred to as “amino acid residue” and “amino acid analog residue”, respectively.
Amino acids are α, β, and γ amino acids, and natural amino acids (in this application, natural amino acids refer to 20 kinds of amino acids contained in proteins. Specifically, Gly, Ala, Ser, Thr, Val, Leu, It is not limited to Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro), and may be an unnatural amino acid. In the case of an α-amino acid, it may be an L-type amino acid, a D-type amino acid, or an α, α-dialkyl amino acid. The selection of the amino acid side chain is not particularly limited, but can be freely selected from, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group in addition to a hydrogen atom. Each may be provided with a substituent, and these substituents are also freely selected from arbitrary functional groups including, for example, N atom, O atom, S atom, B atom, Si atom, and P atom. (That is, an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, cycloalkyl group, etc.).
The “amino acid” and “amino acid analog” constituting the peptide compound include all isotopes corresponding to each. The isotopes of “amino acids” and “amino acid analogs” are those in which at least one atom is replaced with an atom having the same atomic number (number of protons) and a different mass number (sum of protons and neutrons). is there. Examples of isotopes contained in the “amino acid” and “amino acid analog” constituting the peptide compound of the present invention include a hydrogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom, a sulfur atom, a fluorine atom, and a chlorine atom. And 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl, etc. are included.

Examples of the substituent include a halogen-derived substituent such as fluoro (—F), chloro (—Cl), bromo (—Br), iodo (—I) and the like. Examples thereof include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group and the like, which may be substituted with one or more halogens.

Examples of the substituent for forming an ether as the substituent derived from the O atom include an alkoxy group (—OR), and the alkoxy group is an alkylalkoxy group, a cycloalkylalkoxy group, an alkenylalkoxy group, an alkynylalkoxy group, an arylalkoxy group. , A heteroarylalkoxy group, an aralkylalkoxy group, and the like. Examples of the substituent that forms an alcohol moiety include a hydroxyl group (—OH), and examples of the substituent that forms a carbonyl group include a carbonyl group (—C═O—R), and the carbonyl group includes a hydrocarbonyl group ( -C = O-H, an aldehyde is obtained as a compound), an alkylcarbonyl group (a ketone is obtained as a compound), a cycloalkylcarbonyl group, an alkenylcarbonyl group, an alkynylcarbonyl group, an arylcarbonyl group, a heteroarylcarbonyl group , An aralkylcarbonyl group and the like. Examples of the substituent (—CO ₂ H) that forms a carboxylic acid include a carboxyl group, and the substituent that forms an ester group includes an oxycarbonyl group (—O—C═O—R) and a carbonylalkoxy group (—C═ The carbonylalkoxy group includes an alkyloxycarbonyl group, a cycloalkyloxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, an aralkyloxycarbonyl group, and the like. The oxycarbonyl group is selected from alkylcarbonyloxy group, cycloalkylcarbonyloxy group, alkenylcarbonyloxy group, alkynylcarbonyloxy group, arylcarbonyloxy group, heteroarylcarbonyloxy group. Shi group, selected from among an aralkyl carbonyl group.

Examples of the substituent for forming a thioester include a mercaptocarbonyl group (—S—C═O—R) and a carbonylalkyl mercapto group (—C═O—SR), and the like. Selected from alkenylcarbonyl group, mercaptoalkynylcarbonyl group, mercaptoarylcarbonyl group, mercaptoheteroarylcarbonyl group, mercaptoaralkylcarbonyl group, etc., or carbonylalkyl mercapto group, carbonylcycloalkyl mercapto group, carbonylalkenyl mercapto group, carbonyl Alkynyl mercapto group, carbonyl aryl mercapto group, carbonyl heteroaryl mercapto group, carbonyl aralkyl mercapto group It is.

Examples of the substituent that forms an amide group include an aminoalkylcarbonyl group (—NH—CO—R), an aminocycloalkylcarbonyl group, an aminoalkenylcarbonyl group, an aminoalkynylcarbonyl group, an aminocycloalkylcarbonyl group, an aminoarylcarbonyl group, Aminoheteroarylcarbonyl group, aminoaralkylcarbonyl group and the like, or carbonylalkylamino group (—CO—NHR), carbonylcycloalkylamino group, carbonylalkenylamino group, carbonylalkynylamino group, carbonylarylamino group, carbonylhetero An arylamino group, a carbonylaralkylamino group, etc. are mentioned. Further, compounds in which an H atom bonded to an N atom is replaced with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

Examples of the substituent that forms the carbamate group include an aminoalkyl carbamate group (—NH—CO—OR), an aminocycloalkyl carbamate group, an aminoalkenyl carbamate group, an aminoalkynyl carbamate group, an aminocycloalkyl carbamate group, an aminoaryl carbamate group, An aminoheteroaryl carbamate group, an aminoaralkyl carbamate group, etc. are mentioned. Further, compounds in which an H atom bonded to an N atom is replaced with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

Examples of the substituent that forms the sulfonamide group include an aminoalkylsulfonyl group (—NH—SO ₂ —R), an aminocycloalkylsulfonyl group, an aminoalkenylsulfonyl group, an aminoalkynylsulfonyl group, an aminocycloalkylsulfonyl group, an aminoarylsulfonyl Group, aminoheteroarylsulfonyl group, aminoaralkylsulfonyl group, etc., or sulfonylalkylamino group (—SO ₂ —NHR), sulfonylcycloalkylamino group, sulfonylalkenylamino group, sulfonylalkynylamino group, sulfonylarylamino group Sulfonylheteroarylamino group, sulfonylaralkylamino group, and the like. Further, compounds in which the H atom bonded to the N atom is replaced with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group are also included.

Examples of the substituent that forms the sulfamide group include aminoalkylsulfamoyl group (—NH—SO 2 —NHR), aminocycloalkylsulfamoyl group, aminoalkenylsulfamoyl group, aminoalkynylsulfamoyl group, aminocycloalkyl Examples thereof include a sulfamoyl group, an aminoarylsulfamoyl group, an aminoheteroarylsulfamoyl group, and an aminoaralkylsulfamoyl group. In addition, the H atom bonded to the N atom is an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, any two of the same or different, or these form a ring Also included are compounds in which a substituent selected from substituents that may be substituted is substituted.

Examples of the substituent that forms thiocarboxylic acid include a thiocarboxylic acid group (—C (═O) —SH), and the functional group that forms keto acid includes a keto acid group (—C (═O) —CO ₂ H). Is mentioned.

Furthermore, examples of the substituent that forms a thiol group as a substituent derived from an S atom include a thiol group (—SH), such as alkylthiol, cycloalkylthiol, alkenylthiol, alkynylthiol, arylthiol, heteroarylthiol, aralkyl. Thiol is formed. The substituent forming thioether (—S—R) is selected from alkyl mercapto group, cycloalkyl mercapto group, alkenyl mercapto group, alkynyl mercapto group, aryl mercapto group, heteroaryl mercapto group, aralkyl mercapto group, etc. The substituent for forming a sulfoxide group (—S═O—R) includes an alkyl sulfoxide group, a cycloalkyl sulfoxide group, an alkenyl sulfoxide group, an alkynyl sulfoxide group, an aryl sulfoxide group, a heteroaryl sulfoxide group, an aralkyl sulfoxide group, and the like. It is selected from, examples of the substituent to form a sulfone group _{(-S (O) 2 -R)} , alkyl sulfone group, a cycloalkyl sulfone group, alkenyl sulfone group, alkynyl sulfone group, Arirusu Hong group, heteroaryl sulfonic group, selected from among such aralkyl sulfone group, sulfonic acid group (-SO 3 _H) are mentioned as the substituent to form a sulfonic acid.

Examples of the substituent derived from the N atom include an azide group (—N ₃ ), a nitrile group (—CN), and the substituent that forms a primary amine includes an amino group (—NH ₂ ). Examples of the substituent for forming the secondary amine (—NH—R) include an alkylamino group, a cycloalkylamino group, an alkenylamino group, an alkynylamino group, an arylamino group, a heteroarylamino group, an aralkylamino group, and the like. Substituents for forming tertiary amines (—NR (R ′)) include alkyl (aralkyl) amino groups, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, etc. Selected from any two or two of the same substituents, or a substituent that may form a ring Substituent and the like, as a substituent to form an amidino group (-C (= NR) -NR'R " ), an amidino group _{(-C (= NH) -NH 2} ), or a substituent on the N atom An alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and the like, for example, an alkyl (aralkyl) (aryl) amidino group substituted by any three different groups As a substituent for forming a guanidino group (—NR—C (═NR ′ ″) — NR′R ″), a guanidine group (—NH—C (═NH) —NH ₂ ) or R , R ′, R ″, R ′ ″ are any four of the same or different from the alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and aralkyl group, or these Such substituents selected from Good substituent to form a ring.

Examples of the substituent that forms a urea group include an aminocarbamoyl group (—NR—CO—NR′R ″). R, R ′, and R ″ each represent a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, Examples include an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, any three of the same or different ones, or a substituent selected from substituents that may form a ring.

Further, examples of the functional group derived from B atom include alkylborane (—BR (R ′)) and alkoxyborane (—B (OR) (OR ′)). These two substituents are an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, etc. And substituents selected from substituents that may form a ring.

As described above, one or more various functional groups including O atom, N atom, S atom, B atom, P atom, Si atom, and halogen atom, which are usually used in low molecular compounds such as halogen groups, are imparted. May be. That is, one or more further substituents may be added to the alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group represented by one of these substituents. The condition of the functional group satisfying all of these is defined as freely selecting a substituent. In the case of β and γ-amino acids, any steric configuration is allowed as in the case of α-amino acids, and the selection of side chains thereof is not particularly limited, as in the case of α-amino acids. In addition, the amino acid main chain of the amino acid may be free (NH ₂ group) or N-alkylated such as N-methylation (NHR group: R is an optionally substituted alkyl group, alkenyl group, alkynyl) A group, an aryl group, a heteroaryl group, an aralkyl group, or a cycloalkyl group, and a carbon atom from the N atom and a carbon atom from the α-position may form a ring as in the case of proline. Can be freely selected, and examples thereof include a halogen group, an ether group, and a hydroxyl group.

As used herein, “translated amino acid” or “translatable amino acid” refers to an “amino acid” having a side chain that can be translated. As described below, a halogen group, a hydroxyl group (—OH), an alkoxy group (—OR), an ester group (—C (═O) —OR), a thioester group (—C (═O) —SR), Carboxyl group (—CO ₂ H), amide group (—CO—NRR ″ or —NR—CO—R ′), thiol group (—SH), alkylthio group (—SR), sulfoxide group (—S (═O)) -R), sulfone group (-SO ₂ -R), amino group (-NH ₂ ), mono-substituted amino group (-NHR), di-substituted amino group (-NRR '), azido group (-N ₃ ), nitrile An alkyl group, alkenyl group, alkynyl group, aralkyl group, aryl group, heteroaryl group, which may be substituted by one or more groups (—CN), amidino group (—N—C (═N) —NH ₂ ), etc. L having a cycloalkyl group Type α-amino acids, N-methylated L-type α-amino acids, C1-C4 alkyl substitutions such as N-ethylation and N-propylation, and N-aralkyl substituted glycine derivatives such as N-benzylation, , L-type α-amino acids having various highly functional functional groups that can be utilized for substituents having a reaction auxiliary group such as a thiol group, triangular units such as amino groups, and crossing units, and D-tyrosine. Some D-type α-amino acids, β-amino acids such as β-alanine, and α, α-dialkyl amino acids such as α-methyl-alanine (Aib) are also included.

In the present specification, the “drug-like amino acid” is the same skeleton as the “amino acid”, ie, α, β and γ amino acids, and is one of two hydrogen atoms of the main chain amino group (NH ₂ group). And one or two hydrogen atoms of the methylene group (—CH ₂ — group) may be substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or the like. Good. A group in which a hydrogen atom of CH ₂ group is substituted with the above group is defined as a side chain. These substituents include those further substituted with a substituent that functions as a component of a drug-like peptide compound. These are preferably selected from the substituents defined above separately, for example, a hydroxyl group (—OH), an alkoxy group (—OR), an amide group (—NR—CO—R ′ or —CO—NRR ′). ), Sulfone group (—SO2-R), sulfoxide group (—SO—R), halogen group, hydroxylamino group (—NR—OR ′), aminohydroxy group (—O—NRR ′), etc. Optionally substituted by one or more substituents. Any of those corresponding to L-type amino acid, D-type amino acid, α, α-dialkylamino acid may be used. Drug-like amino acids need not necessarily be translatable. Side-chain part of the peptide obtained from “translated amino acid” (for example, when hit compound is obtained with D-tyrosine, hit compound is obtained with β-alanine, a D-type amino acid chemically modified therefrom. In this case, it includes β-amino acids chemically modified therefrom, or all amino acids that can be chemically synthesized by optimizing the structure of N-substituted moieties by chemical conversion of N-methyl amino acids. Since these amino acids function as components of a drug-like peptide compound, they are selected from a range in which the peptide compound obtained by chemical modification performed after translation becomes drug-like. As described below, for example, lysine having an aminoalkyl group is not included in the drug-like amino acid when the amino group does not participate in post-translational modification. However, when the amino group of lysine is used as a reactive functional group for post-translational modification (for example, a crossing unit), the lysine unit is included as a drug-like amino acid unit. Thus, whether or not it falls under “drug-like amino acid” is determined by the functional group after conversion by post-translational modification. Examples of such substituents may include, for example, an ester group (—CO—OR), a thioester group (—CO—SR), a thiol group (—SH), or a protection group among the substituents defined above. Thiol group, amino group (—NH 2), mono-substituted amino group (—NH—R) or di-substituted amino group (—NRR ′), protected amino group, substituted sulfonylamino group (—NH—SO 2 —) R), an alkylborane group (—BRR ′), an alkoxyborane group (—B (OR) (OR ′)), an azide group (—N3), a keto acid group (—CO—CO2H), a thiocarboxylic acid group (—CO -SH), phosphoryl ester group (-CO-PO (R) (R ')), acylhydroxylamino group (-NH-O-CO-R) and the like.

The amino acid analog of the present invention preferably means α-hydroxycarboxylic acid. The side chain of α-hydroxycarboxylic acid may have various substituents in addition to hydrogen atoms as in the case of amino acids (may have free substituents). The steric structure of the α-hydroxycarboxylic acid may correspond to the L-type or D-type of amino acid, and there is no particular restriction on the selection of the side chain. , An alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, and the like. The number of substituents is not limited to one and may be two or more. For example, it may have an S atom and further have a functional group such as an amino group or a halogen group.

As used herein, “translated amino acid analog” or “translatable amino acid analog” means an amino acid analog that can be translated among “amino acid analogs”. Specifically, for example, a compound in which the main chain amino group of the L-type amino acid is replaced with a hydroxyl group can be mentioned. Examples include L-lactic acid, α-hydroxyacetic acid, L or D-phenyllactic acid. The “drug-like amino acid analog” is not particularly limited as long as it functions as a component of a drug-like peptide compound among the “amino acid analogs”. This range is the same as the definition of the side chain or N-substituted moiety of the drug-like amino acid described above. Specifically, for example, L or D-lactic acid, or various drug-like substituents (for example, halogen group, hydroxyl group, optionally substituted alkyl group, alkenyl group, alkynyl group) on the side chain methyl group , A cycloalkyl group, an aralkyl group, an aryl group, a heteroaryl group, etc.), α-hydroxyacetic acid, L or D-phenyllactic acid, or various drug-like substituents on the side chain benzyl group (for example, , Halogen groups, hydroxyl groups, optionally substituted alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aralkyl groups, aryl groups, heteroaryl groups, etc.). In addition, a drug-like amino acid analog is not necessarily translatable. When a peptide compound becomes drug-like by chemical modification after translation of the “translated amino acid analogue”, the amino acid analogue is also included in the “drug-like amino acid analogue”. Examples of such amino acid analogs include α-hydroxycarboxylic acids having an SH group attached to the side chain and α-hydroxycarboxylic acids having an amino group or a protected amine moiety attached to the side chain. . For example, the SH group can be removed by desulfurization after post-translational modification, and the amino group can be converted to amide or the like by post-translational modification. Specific examples include R-2-hydroxy-3-sulfanylpropanoic acid.

The N-terminal carboxylic acid analog of the present invention is a compound having an amino group and a carboxyl group at the same time and having 3 or more atoms between them, various carboxylic acid derivatives having no amino group, It may be a peptide formed from 4 residues, or an amino acid whose main chain amino group is chemically modified by an amide bond with a carboxylic acid. Moreover, you may have a boric acid and boric-ester part which can be used for the cyclization in a curve part. Moreover, the carboxylic acid which has a double bond site | part and a triple bond site | part may be sufficient, and the carboxylic acid which has a ketone and a halide may be sufficient. In addition, the portion other than the functional group that defines these compounds is also widely selected from alkyl groups, aralkyl groups, aryl groups, cycloalkyl groups, heteroaryl groups, alkenyl groups, alkynyl groups, etc. that may be substituted (free Substituents).

As used herein, “translated N-terminal carboxylic acid analog” or “translatable N-terminal carboxylic acid analog” refers to an N-terminal carboxylic acid analog that can be translated among “N-terminal carboxylic acid analogs”. Means the body. Specifically, for example, a compound in which a double bond and a carboxylic acid are connected by an alkyl group (but-3-enoic acid, penta-4-enoic acid, etc.), L having an N-terminal amidated by acetylation or the like Type amino acids (Ac-Phe, Ac-Ala, Ac-Leu, etc.), α-hydroxycarboxylic acid derivatives in which the OH group is alkylated, dipeptides, tripeptides and the like. The “drug-like N-terminal carboxylic acid analog” is not particularly limited as long as it functions as a component of a drug-like peptide compound among the “N-terminal carboxylic acid analogs”. These substituents include the same substituents as defined in the side chain of the drug-like amino acid. Specifically, for example, in a compound in which a double bond and a carboxylic acid are connected by an alkyl group (but-3-enoic acid, penta-4-enoic acid, etc.), the substituent is within a drug-like range from the carbon atom. Among the L-type amino acids (Ac-Phe, Ac-Ala, Ac-Leu, etc.) in which the N-terminal is amidated by acetylation or the like, the acetyl group, the side chain or the α-position hydrogen atom is dragged Among the compounds substituted in the like range, α-hydroxycarboxylic acid derivatives in which the OH group is alkylated, the alkyl group of the OH group, the side chain of the hydroxycarboxylic acid, the α-position hydrogen atom, etc. Examples include substituted compounds, dipeptides substituted in a drug-like range, and tripeptides. In addition, a drug-like N-terminal carboxylic acid analog does not necessarily need to be translatable. When the peptide compound becomes drug-like by translating the “translated N-terminal carboxylic acid analog” after translation, the N-terminal carboxylic acid analog is also included in the “drug-like N-terminal carboxylic acid analog”. It is. Examples of such N-terminal carboxylic acid analogs include dipeptides with an amino group remaining at the N-terminus, γ-aminocarboxylic acid, and δ-aminocarboxylic acid.

Membrane permeability of peptide compound In order for the peptide compound of the present invention to have good membrane permeability, the total number of amino acids contained in the peptide compound is preferably 13 or less.
There is no particular restriction on the selection of each unit (amino acid residue), but the CLogP (computer-based) of the molecule formed is reconsidered in the form of the molecule (main chain structure) in which all post-translational chemical modifications have been completed. It is preferred to select such that the calculated partition coefficient (calculated using Daylight Version 4.9 of Daylight Chemical Information Systems, Inc.) exceeds 6. In order to ensure particularly good membrane permeability, it is preferable to select CLogP so that it exceeds 8 and does not exceed 15.

In order to ensure membrane permeability, the amino acid side chain is more preferably selected from drug-like substituents. For example, an optionally substituted alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aralkyl group, aryl group, heteroaryl group, for example, a halogen group, a hydroxyl group (—OH), an amide group ( -CO-NRR 'or -NR-CO-R'), a sulfone group (-SO ₂ -R), an ether group (-OR), or the like is preferred (R and R 'are similarly substituted by these) An alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, which may be substituted). Examples of the aryl group part of the aryl group and aralkyl group include a phenyl group, a basic group such as a pyridine group, two or more heteroatom-containing groups such as a thiazole group, a hydrogen atom donor such as an imidazole group, and an indole group. Fused aromatic rings are also acceptable.

On the other hand, examples of polar functional groups that are not preferable for obtaining membrane permeability include alkylamino groups and alkylguanidine groups that are extremely ionized in the living body (around pH = 7). It is preferable that the functional group is not contained.

For the purpose of obtaining membrane permeability, N-alkylated compounds such as N-methylated or cyclized with α-position carbon atoms such as proline may be used as amino acids or amino acid analogs constituting peptide compounds. Well, it is preferable that the number is 2 or more per peptide molecule. Further, it is preferable that at least one amide bond that is not N-alkylated is present per peptide molecule. Desirably, there are three or more N-alkylated ones and three or more non-N-alkylated one per peptide molecule. This N-alkylation includes all chemical modifications other than NH and may therefore be substituted (selection of substituents is similar to amino acid side chain substituents to ensure membrane permeability above) It is selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups. N-alkylation includes those that form a ring structure between the N atom and the α-carbon, such as proline.

It is more preferable that the C-terminal part of the peptide compound is not a carboxylic acid but is chemically modified. For example, an optionally substituted alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aryl group, heteroaryl group, aralkyl group amide compound (—CO—NRR) such as piperidine amide obtained by reacting a carboxylic acid site with piperidine, etc. ': One of R and R' may be a hydrogen atom, the other may be chemically modified, both R and R 'may be chemically modified, and R and R' form a ring, for example, It may be chemically modified such as piperidine amide, R and R ′ may be both hydrogen atoms, or a carboxylic acid group may be substituted with a methyl group or a trifluoromethyl group. It is preferably converted into various nonionic functional groups such as a group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group. The selection of the substituent is the same as the substituent on the amino acid side chain for ensuring the membrane permeability above. Moreover, the amino acid which comprises a peptide compound can also optimize the compound synthesize | combined by translation. In addition, amino acids constituting the obtained peptide compound are not limited to those synthesized by translation.

Here, optimization means chemical modification by converting the structure of each amino acid in a compound synthesized by translation from “translated amino acids” to become more drug-like peptide compounds. This means that the peptide compound is chemically modified to have a peptide and / or chemically modified to be a peptide compound in which toxicity is further avoided. Here, as preferable toxicities to be avoided or avoided, for example, hERG inhibition (heart toxicity), AMES test (carcinogenicity test), CYP inhibition (drug interaction test), CYP induction, GSH Binding ability measurement test (covalent bond formation test of peptide or peptide metabolite with glutathione) and the like. In these tests, there may be cases where both the peptide compound itself and its metabolite are involved, and in order to ensure a negative result in the AMES test, CYP induction, GSH binding ability measurement test, in particular, covalent binding It is preferred to avoid metabolites that can produce. For this reason, the cyclization sites contained in all peptide compounds in the display library do not form such potential functional groups, and cyclization is performed with a cyclization reaction that has a certain degree of stability against oxidation reactions. It is preferred that For example, as practiced in many low molecular weight compounds, when phenylalanine is identified as a translated amino acid, various chemical modifications such as alkyl substitution and halogen substitution can be performed on the phenyl group. Such conversion is greatly different from conventional chemical modification by N-methylation or cyclization of natural peptides in that it can be carried out without greatly impairing the three-dimensional structure of the translationally synthesized compound. Furthermore, in many cases, naturally-occurring peptides often have extremely ionized arginine and lysine residues contributing to the activity, so that it is difficult to easily convert them into drug-like functional groups. By using this technology, functional groups previously limited only by drug-like functional groups have acquired activity against medicinal targets and are cyclized by drug-like cyclization methods. With this chemical modification, clinical candidate compounds can be obtained with the same accuracy and success rate as small molecule chemistry. On the other hand, the CLogP value, which is an index of fat solubility, can be easily adjusted during the optimization process. Moreover, the binding activity to the target can be enhanced by alkylation or halogenation, which is usually performed even in the optimization of low molecular weight compounds. Usually, an improvement in activity of 10 to 50 times can be expected. These normal conversions can also increase the CLogP value at the same time. For example, it is possible to increase about 0.7 by chlorination and about 0.5 by methylation. If it has extremely ionized functional groups, increasing the CLogP value during the optimization process is not sufficient to obtain excellent membrane permeability, but if it does not have extremely ionized functional groups, A CLogP value sufficient for membrane permeation can be obtained during the optimization process. Therefore, for example, when a hit compound with a CLogP value of 5 is obtained from a display library with an average CLogP value of around 6, the CLogP value is further increased to a range of 8 to 15 which is optimal for membrane permeation. It can be judged that optimization is sufficiently possible

Confirmation of membrane permeability of the peptide compound of the present invention can be achieved by known methods such as rat intestinal tract method, cultured cell (Caco-2, MDCK, HT-29, LLC-PK1, etc.) monolayer method, Immobilized Artificial Membrane chromatography (Immobilized artificial membrane chromatography) method, method using a partition coefficient, ribosome membrane method, PAMPA (Parallel Artificial Membrane Permeation Assay) method and the like can be used. Specifically, for example, when the PAMPA method is used, the literature by Holger Fischer et al. .Sci. 2007, 31, 32-42). More specifically, it can be confirmed according to the method described in Example 19-2.

When the membrane permeability of hydrochlorodiazide, furosemide and metoprolol having membrane permeability as an oral pharmaceutical is measured by the PAMPA method, the iPAMPA Pe values are 0.6 × 10 ⁻⁶ , 1.5 × 10 ⁻⁶ and 2.9 × 10 ⁻⁵ , respectively. The membrane permeability of the peptide compound of the present invention, for example, was able to obtain membrane permeability that can be used as a pharmaceutical when the value of iPAMPA Pe when measured by the PAMPA method is usually 1.0 x 10 ^-6 or more. it is possible to think, in is preferably 1.0x 10 ^-6 or more, more preferably 1.0 × 10 ^-5 or more, more preferably particularly not less 1.5 × 10 ^-5 or more, 2.0x10 ^-5 or higher Even more preferably.

-Metabolic stability of peptide compound In order for the peptide compound of the present invention to have good metabolic stability, the total number of amino acids contained in the peptide compound is preferably 9 or more, more preferably 11 or more. . If the total number of amino acids is a peptide compound of 9 or more, the above-described conditions for having membrane permeability do not affect the metabolic stability of the peptide compound.

The metabolic stability of the peptide compound of the present invention can be confirmed using a known method, for example, hepatocytes, small intestinal cells, liver microsomes, small intestine microsomes, liver S9 and the like. Specifically, for example, the stability of peptide compounds in liver microsomes is described in LL von Moltke et al. (Midazolam hydroxylation by human liver microsomes in vitro: inhibition by fluoxetine, norfluoxetine, and by azole antifungal agents. J Clin Pharmacol , 36 (9), 783-791). More specifically, it can be confirmed according to the method described in Example 18-2.

Metabolic stability is determined when the value of liver clearance (CLh ソ ー ム int (μL / min / mg protein)) is 150 or less when the stability in liver microsomes is measured according to the above method. It can be considered that metabolic stability that can be used as a pharmaceutical product has been obtained, and is preferably 100 or less. In the case of drugs metabolized by CYP3A4, in order to avoid small intestine metabolism in humans, 78 or less (non-patent literature: M. Kato et al. The intestinal first-pass metabolism of substances of CYP3A4 and P-glycoprotein-quantitative analysis based on information from the literature. Drug Metab. Pharmacokinet. 2003, 18 (6), 365-372.) In order to show bioavailability of about 30% or more in humans, 35 or less (FaFg = 1, It is more preferable that the protein binding rate is 0%).

Scheme A

Method for Producing Peptide Compound Having Cyclic Portion The peptide compound having a cyclic portion of the present invention can be produced using the method described below.
For example, the manufacturing method containing the following manufacturing methods can be mentioned.
1) An acyclic peptide compound composed of an amino acid residue and / or an amino acid analog residue, or an amino acid residue and / or an amino acid analog residue and an N-terminal carboxylic acid analog is encoded with the peptide compound A process of translating and synthesizing from the nucleic acid
The acyclic peptide compound comprises an amino acid residue or amino acid analog residue having a reactive site on one of the side chains on the C-terminal side, and an amino acid residue or amino acid analog having another reactive site on the N-terminal side. A body residue or an N-terminal carboxylic acid analog;
2) Combine the reaction point of the amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog on the N-terminal side with the reaction point of the amino acid residue or amino acid analog residue in the side chain on the C-terminal side And forming an amide bond or a carbon-carbon bond.
A known method can be used for the translational synthesis of the present invention.

N-terminal introduction of a translatable amino acid, a translatable amino acid analog or a translatable N-terminal carboxylic acid analog is generally translated with methionine as the N-terminal amino acid as the translation initiation amino acid, but other desired It is also possible to use a method in which the N-terminus is translated as a desired amino acid by translating the amino acid with an aminoacylated translation initiation tRNA. It is known that the introduction of an N-terminal amino acid has an amino acid tolerance higher than that at the time of extension, and uses amino acids and amino acid analogs that are significantly different in structure from natural amino acids (non-patent literature: J Am Chem Soc 2009 Apr 15; 131 (14): 5040-1. Translation initiation with initiator tRNA charged with exotic peptides. Goto Y, Suga H.). For example, in the present invention, there are the following methods for introducing a translatable amino acid other than methionine, a translatable amino acid analog, or a translatable N-terminal carboxylic acid derivative into the N-terminus. As a translation initiation tRNA, a tRNA acylated with a triangle unit is added to a translation system from which methionine, formyl donor, or methionyl transferase has been removed, and the translation initiation codon (for example, ATG) is encoded and translated to translate the end unit. A peptide compound or peptide compound library before cyclization as a triangular unit is constructed. When various combinations of a translation initiation tRNA whose anticodon is not CAU and a codon corresponding to the anticodon are used as a combination of the translation initiation tRNA and the initiation codon, diversity can be provided at the N-terminus. In other words, a desired amino acid, amino acid analog or N-terminal carboxylic acid analog is aminoacylated to multiple types of translation initiation tRNAs having different anticodons, and an mRNA or mRNA library having the corresponding codon as the start codon is translated. Thus, the peptide compound before cyclization or a library of peptide compounds can be prepared in which the N-terminal residue is not limited to one type. Specifically, for example, Mayer C, et al. Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity in initiation. Biochemistry. 2003, 42, 4787-99. (Initial tRNA mutations with anticodons other than CAU start translation from amino acids other than f-Met with E. coli and expression of proteins containing the same codons before cyclization using the method described in 4787-99.) Peptide compounds or peptide compound libraries can be generated.

Also, the reaction point of the amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog on the N-terminal side and the reaction point of the amino acid residue or amino acid analog residue in the side chain on the C-terminal side are combined. For example, the amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog having an amino group and a reaction auxiliary group in the N-terminal side chain, and an amino acid having an active ester group in the side chain A method of obtaining a cyclic compound by amide-bonding a residue or amino acid analog residue, having an amino group at the N-terminal amino acid residue, amino acid analog residue or N-terminal carboxylic acid analog, and an active ester group A method for obtaining a cyclic compound by amide-bonding an amino acid residue or amino acid residue in a side chain, an N-terminal amino acid residue, an N-terminal amino acid analog residue, or a reaction point of an N-terminal carboxylic acid analog and the side One in the chain Carbon and the reaction point of the amino acid residues or amino acid analog having a reactive point - like way to carbon bond. Further, the present invention is not limited to the above example, and for example, an amino acid residue on the N-terminal side, an amino acid analog on the N-terminal side, or an active ester group on the N-terminal carboxylic acid side, and an amino group on the side chain on the C-terminal side A functional group arrangement opposite to the above may be used, such as amide bonding (which may have a reaction auxiliary group).

The following describes the reaction design taking the case of using amide cyclization as an example. It may be necessary to improve the reactivity of the amine to be reacted with respect to other functional groups in order to obtain reaction selectivity with the basic functional group that is not desired to react with the amino group to be reacted with the triangular unit. possible. In general, as a method of activation from an amine, for example, a reaction auxiliary group can be introduced in the vicinity of the amine. The reaction auxiliary group is not particularly limited as long as it can activate the amine to such an extent that RNA does not react. For example, a mercaptoethyl group or a mercaptopropyl group can be introduced from a terminal amine, and cysteine or the like can be used. It is also possible to position the thiol moiety from the α-position of the amine (see Scheme C). These thiol groups may or may not be protected during the translation introduction process, but a thiol group is generated by a deprotection reaction at the time of reaction or in advance as necessary. The reaction of the amine thus activated with the carboxylic acid active ester gives an amide-cyclized peptide at the desired position. RNA to which the SH group of the obtained cyclic peptide is added with a reagent such as TCEP (tris (2-carboxylethyl) phosphine) and VA-044 (2,2′-azobis-2- (2-imidazolin-2-yl) propane) Can be desulfurized under mild reaction conditions where no reaction occurs.

When carrying out a display library by translation, a thioester is preferred as the active ester of the crossing unit to be translated. When an SH group is used as the reaction auxiliary group, for example, the following combinations are performed. When translational synthesis of the SH group in an unprotected state, it is preferable to use an aspartic acid derivative as the thioester that is a crossing unit. In this case, all the black circle units and the square units can be arbitrarily selected from translated amino acids and translated amino acid analogs. On the other hand, when translation synthesis is carried out with the SH group having a protecting group, the square unit on the C-terminal side of the aspartic acid thioester is an N-alkylated amino acid (such as proline or N-methyl alanine). Is preferably selected from.

Scheme C

As a general formula, an amino acid having a reaction auxiliary group in the vicinity of an amine as a triangular unit, an N-terminal carboxylic acid analog, for example, an N-terminal amino acid can be written as compound N-1 or N-2. Substituents of these compounds N-1 and N-2 are preferably defined as described above, among the substituents represented by R, except for protecting groups (R1, R23, trityl group, etc.) This is the same as the definition of the side chain of the drug-like amino acid. In addition, compounds in which the compounds obtained as a result of their introduction can be synthesized by translation are more preferred, but those in which the analog is translated and synthesized even if the derivative itself is not translated and synthesized are included (see paragraphs described later). (See, for example, the next three paragraphs).

R1 is selected from among the protecting groups of SH group represented by a hydrogen atom or the like, or S-R23 group, or C _{(Phe) 3} group (trityl group). R23 is an alkyl group such as a methyl group, an ethyl group, an isopropyl group or a tert-butyl group, an aryl group such as a phenyl group, a p-trifluoromethylphenyl group or a p-fluorophenyl group, an aralkyl group such as a benzyl group or a phenethyl group. Selected from a group, a heteroaryl group, an alkenyl group, an alkynyl group, and the like. These groups are selected from substituents that enable the resulting compound N-1 or N-2 to be translationally synthesized. For example, an N, N-dimethylaminoethyl mercapto group in which the ethyl group of R23 is substituted with a dimethylamino group may be used. When R1 has a protecting group (other than H atom), the protecting group selected is selected from protecting groups capable of translational synthesis, and deprotected in a translation synthesis solution, and cyclized. If it becomes a hydrogen atom before making it, it will not specifically limit and can be selected. Since protecting groups such as the S—R23 group are slowly deprotected in the translation synthesis solution, they can be deprotected without the need to positively define separate deprotection conditions. If necessary, a deprotecting agent can be added under various reaction conditions described in the present specification. (Description of protecting group of SH group.)

R2 and R3 are defined similarly to the definition of the side chain of a drug-like amino acid. For example, R2 and R3 preferably represent a hydrogen atom, an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group, or R2 And R3 represents a substituent that forms a ring, or R2 or R3 represents a substituent that forms a ring with R4. More preferably, it is selected from a hydrogen atom, a C1-C4 alkyl group which may be substituted with a C1-C4 alkyl group, an alkoxy group, a halogen group or the like. In addition, the R3 group can accept both L-type amino acids and D-type amino acids. Preferably, the steric structure of the R3 group corresponds to the L-type amino acid when the R3 group is assumed to be a hydrogen atom. *

R4 is a unit that links an S (sulfur) atom and an amino acid site. The typical structure is shown below. Both units may be substituted methylene group (partial structure N-3, C1 unit), may be substituted ethylene group (partial structure N-4, C2 unit), may be substituted propylene group (partial structure) N1 and C6 units such as N-5 and C3 units). Examples of the substituent in the methylene group, ethylene group and propylene group which may be substituted include, for example, the compound N-1 in which R13 is methylated (R14 = H), and dimethyl such as R13 = R14 = Me. Compound N-1 and the like. Of these, when any of the derivatives is translationally synthesized, all other derivatives are included in this definition even if they are not translationally synthesized. For example, when R13 = R14 = H is translated and synthesized, a derivative such as R13 = R14 = Me is included in the definition of the drug-like amino acid side chain even if it is not translated and synthesized. Included as a triangular unit. R13, R14, R15, R16, R17, R18, R19, R20, R21, R22 and the like have the same definition as R4, but are substituted with, for example, a hydrogen atom, a C1-C4 alkyl group, an alkoxy group, a halogen atom, etc. Selected from C1-C4 alkyl groups which may be substituted. A cyclized structure may be formed between them. Particularly preferably, it is selected from a hydrogen atom and a methyl group. It can also be directly linked from the aryl carbon of the aromatic compound (partial structure N-6). Further, they can be linked with an aralkyl structure (partial structures N-7 and N-8). In the partial structure N-7, any of the divalent groups may be on the nitrogen atom side or on the sulfur atom side. In the following scheme, the connecting position is limited to ortho, but not limited to ortho, meta, para, etc. are possible. Although an aryl group is represented by a phenyl group, the phenyl group may be substituted with a substituent such as a halogen group, an alkoxy group, or a trifluoromethyl group, and an aryl group other than a phenyl group (that is, a heteroaryl group is included) Various aromatic rings) may be used.

R11 and R12 are also selected from the same partial structure as R4. For example, it can be selected from the partial structures N-3, N-4, N-5, N-6, N-7, and N-8. R12 can also contain a C0 unit (when the linking site is directly attached).

Preferred structural formulas of Compound Structure N-1 and Compound Structure N-2 are shown in Compound Structures N-9, N-10, N-11, and N-12. In compound N-9, the R12 site of compound N-1 is a C0 unit and the linking site is directly linked. In compound N-10, the R11 site of compound N-2 is linked by a C1 unit (corresponding to partial structure N-3). In compound N-11, the R11 site of compound N-2 is linked by a C2 unit (corresponding to partial structure N-4). In compound N-12, the R11 site of compound N-2 is linked by a C3 unit (corresponding to partial bond N-5).

R5 to R10 are the same as defined above for R13 to R18.

Further, these desirable structural formulas are shown in the following compounds N-13, N-14, N-15, N-16, N-17, N-18, N-19, and N-20. In compound N-13, the R4 site of compound N-9 is linked by a C1 unit (corresponding to partial structure N-3). In compound N-14, the R4 site of compound N-10 is linked by a C2 unit (corresponding to partial structure N-4). In compound N-15, the R4 site of compound N-11 is linked by a C2 unit (corresponding to partial structure N-4). In compound N-16, the R4 site of compound N-12 is linked by a C2 unit (corresponding to partial structure N-4). In compound N-17, the R4 site of compound N-9 is linked by a C2 unit (partial structure N-4). In compound N-18, the R4 site of compound N-10 is linked by a C3 unit (partial structure N-5). In compound N-19, the R4 site of compound N-11 is linked by a C3 unit (partial structure N-5). In compound N-20, the R4 site of compound N-12 is linked by a C3 unit (partial structure N-5).

Although the chemical structures that can be represented by the general formula compounds N-1 and N-2 have been described so far, the definition of the triangular unit containing the reaction auxiliary group SH is not limited thereto. That is, it has an amino group which is a unit to be reacted with the crossing unit and a reaction auxiliary group, and is selected from arbitrary structures among structures capable of translational synthesis. The amino group may be derived from a main chain or a side chain. The triangular unit is not necessarily present at the N-terminal, and a square unit (straight chain part) may be present on the N-terminal side of the triangular unit. A chemical structure in which the positional relationship between the SH group and the amino group is in the range of 2 to 6 linking atoms such as β (two linking atoms between two functional groups), γ (three linking atoms) is preferable. More preferably, the positional relationship between the SH group and the amino group is β or γ. Moreover, even if the unit which has an active ester functional group is arrange | positioned at the triangular unit, the unit containing an amine may be arrange | positioned at the crossing unit side.

As a general formula of an amino acid residue having an active ester group in the side chain, it can be expressed as compound C-1. Preferably, the substituents excluding these active ester sites are the same as the definition of the side chain of the drug-like amino acid defined above. Even if the derivative itself is not translationally synthesized, it includes those in which the analog is translated and synthesized. In the present application, an active ester means a carboxylic acid derivative that can be reacted with an amino group site directly or via a reaction auxiliary group, and is not particularly limited as long as it is an active ester or an active thioester having such properties. To do. R25 is selected from a hydrogen atom or an active ester group. The active ester is represented by, for example, N-hydroxysuccinimide (ONSu) group, OAt group, OBt group, methylthioester, arylthioester, aralkylthioester and the like as widely used. These active ester moieties are generally used as compound substituents (for example, halogen groups, nitro groups, trifluoromethyl groups, nitrile groups, etc., which are often used for the purpose of increasing reactivity). Electron-withdrawing groups, alkoxy groups such as methoxy groups, which are often used for the purpose of lowering the reaction and increasing the reaction selectivity, electron-donating groups such as alkyl groups such as methyl groups, t-butyl groups and isopropyl Examples include bulky substituents typified by groups, sulfonic acid groups, disubstituted amino groups such as dimethylamino groups, etc., taking into consideration the affinity with water after being carried out in water. Examples include high-fat-soluble groups such as long-chain alkyl groups in consideration of affinity with lipophilicity.) All derivatives having similar reactivity are also included.

R2 and R3 are as defined above for the amine moiety.

R26 is the same as the definition of R4, and its typical structure is shown below. Both units can be connected by C1 to C6 units such as methylene group (partial structure N-3), ethylene group (partial structure N-4), propylene group (partial structure N-5) and the like. In R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, these substituents are preferably the same as the definition of the side chain of the drug-like amino acid defined above. Even if the derivative itself is not translationally synthesized, it includes those in which the analog is translated and synthesized. For example, it is selected from a hydrogen atom, a C1-C4 alkyl group, a C1-C4 alkyl group which may be substituted with a halogen atom and the like. A cyclized structure may be formed between them. More preferably, it is selected from a C4 unit, a C5 unit and a C-6 unit in addition to a methylene group (C1 unit, partial structure N-3). More preferably, a C1 unit (partial structure N-3) is selected. It can also be directly linked from the aryl carbon of the aromatic compound (partial structure N-6). Further, they can be linked with an aralkyl structure (partial structures N-7 and N-8). In the following scheme, the connecting position is limited to ortho, but not limited to ortho, meta, para, etc. are possible. Although the phenyl group is shown as the aryl group, the phenyl group may be substituted with a substituent such as a halogen group or an alkoxy group, or an aryl group other than the phenyl group may be used.

Among compounds C-1, a preferred structure is shown in compound C-2. R27 represents a hydrogen atom, an alkyl group that may be substituted, an alkenyl group that may be substituted, an alkynyl group that may be substituted, an aryl group that may be substituted, a heteroaryl group that may be substituted, Or an aralkyl group to which an optionally substituted alkyl group may be provided. These substituents are not particularly limited as long as compound C-2 obtained as a result of selection of the substituent can be translationally synthesized. For example, as a substituent, an electron withdrawing group such as a halogen group, a nitro group, a trifluoromethyl group, or a nitrile group, which is often used for the purpose of increasing the reactivity, or a purpose of increasing the reaction selectivity by lowering the reaction Alkoxy groups such as methoxy groups, electron donating groups such as alkyl groups such as methyl groups, bulky substituents typified by t-butyl groups and isopropyl groups, etc. In addition, a sulfonic acid group considering the affinity with water, a disubstituted amino group such as a dimethylamino group, and a highly lipophilic group such as a long-chain alkyl group considering lipophilicity are selected. Preferably, it is selected from an optionally substituted alkyl group, an optionally substituted cycloalkyl group, and an optionally substituted aralkyl group. More preferably, it is selected from an alkyl group and an aralkyl group in which the aryl moiety may be substituted.
R3 is defined similarly to the definition of the side chain of a drug-like amino acid, and is selected from, for example, a C1-C4 alkyl group, a C1-C4 alkyl group which may be substituted with a halogen, etc., and particularly preferably a hydrogen atom . In addition, although the R3 group conforms to both L-type and D-type amino acids when the R3 group is assumed to be a hydrogen atom, those corresponding to L-type amino acids are preferred.

A more preferred structure is shown in Compound C-3. R28 and R29 are each defined similarly to the definition of the side chain of a drug-like amino acid, but for example, a hydrogen atom, an optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, a substituted An optionally substituted C2-C6 alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted C1-C6 alkyl group optionally substituted aralkyl group, substituted It is selected from cycloalkyl groups that may be present. Examples of these substituents include monomethylation (R28 = Me, R29 = H), dimethylation (R28 = R29 = Me), monotrifluoromethylation (R28 = CF3, R29 = H), and the like.

R3 is selected from a hydrogen atom, a C1-C4 alkyl group, a C1-C4 alkyl group optionally substituted with halogen, etc., and a hydrogen atom is particularly preferred. In addition, although the R3 group conforms to both L-type and D-type amino acids when the R3 group is assumed to be a hydrogen atom, those corresponding to L-type amino acids are preferred.
Similarly to Compound C-1, Compound C-2, and Compound C-3, the compound COH-1, Compound COH-2, and Compound COH-3 can also be selected.

When Compound C-2 or Compound C-3 is used, the reaction with Compound N-1 or Compound N-2 can be allowed to proceed gently and selectively. The reaction can proceed smoothly even in a translation solution (for example, 37 ° C. and pH around 7.3). Removal of the reaction auxiliary group can be easily performed under reaction conditions in which RNA is stable.

When an active ester is arranged on the N-terminal side (triangular unit), an amine unit having a reaction auxiliary group may be arranged on the C-terminal side (crossing unit). In this case, an amino group and a thiol group are arranged in the side chain of the intersection unit. These may be protected at the translation stage, but are deprotected immediately before the reaction. The amino group and the thiol group are not particularly limited as long as they are present in the vicinity, but the relationship between the two is preferably the β-position or γ-position.

That is, a drug-like ring is formed by an amide condensation reaction with an active ester such as a thioester on the side chain of the triangular unit and an amino group of the side chain (having a reaction auxiliary group such as thiol in the vicinity) on the other (crossing unit). All the techniques for making them into are also included in the present application, and either functional group may be arranged in a triangular unit or a crossing unit.

Hereinafter, specific examples of structures different from those of Compound N-1 and Compound N-2 are shown as amine sites having reaction auxiliary groups. In either case, the amino group and thiol group may be protected as necessary. The method described herein can be used to select the protecting group and the deprotection reaction conditions. For compound Na-10, as shown in the figure, a structure in which two carbon atoms are arranged between an amino group and a thiol group can be selected. For compound Na-11, as shown in the figure, a structure in which three carbon atoms are arranged between an amino group and a thiol group can be selected. A Na-7 group, Na-8 group or Na-9 group is arranged as a substituent in any of Ra20 to Ra25. The definition of Ra7 is as described above. However, only in the case of Compound Na-10 or Compound Na-11, Ra-7 can be selected from Na-7, Na-8, and Na-9 groups. . The Na-7 group, Na-8 group and Na-9 group are selected by being limited to only one of Ra-7 or Ra20 to Ra25. Ra20 to Ra25 other than the substituent in which the Na-7 group, Na-8 group, or Na-9 group is selected include a hydrogen atom, an alkyl group that may be substituted with a drug-like functional group, an aryl group, A heteroaryl group, an aralkyl group, and the like are selected from. Preferably, it is selected from a hydrogen atom and an alkyl group.

When a unit having an active ester such as a thioester group is selected on the triangular unit (N-terminal side), these include, for example, Compound C-1, Compound C-2, Compound C-3, Compound Ca-1, Compound COH -1, compound COH-2, and compound COH-3. Compound C-1 and the like retain the main chain amino group after cyclization, whereas compound Ca-1, compound COH-1, compound COH-2, and compound COH-3 have no amino group. Therefore, it is more preferable because it becomes more like a drug. In this case, the crossing unit is selected from compound Na-10 (Na-7 group or Na-8 group), compound Na-11 (Na-7 group or Na-8 group), and the like. These compounds may be translated in a protected state. The protecting group that can be translated and the deprotection conditions under reaction conditions stable to RNA can use the methods described herein. More preferably, the use of the compound Na-7 group is more preferable than the use of the Na-8 group because of higher metabolic stability.

Also, Compound C-1, Compound C-2, Compound C-3, Compound COH-1, Compound COH-2, Compound COH-3, etc. having an active ester group at the intersection unit (C-terminal side) were selected. In this case, the triangular unit (on the N-terminal side) can be selected from Compound Na-10 and Compound Na-11 in addition to Compound N-1 and Compound N-2 already described. When the triangular unit is at the N-terminus, in addition to compound N-1 and compound N-2, compound Na-10 (Na-8 group and Na-9 group) or Na-11 (Na-8 group, And Na-9 groups) are preferred. This is because when the Na-7 group is used, the main chain amine is retained and the drug likeness is lowered. On the other hand, when the triangular unit is not N-terminal, compound Na-10 (Na-7 group and Na-8 group) and compound Na-11 (Na-7 group and Na-8 group) can be used. . In this case, it is more preferable to use an amino acid derivative or an N-terminal carboxylic acid derivative at the N-terminus. This is because the main chain amino group is not retained at the N-terminus.

Further, what is imparted to the compound Na-10 and the compound Na-11 is not limited to the Na-7 group, Na-8 group, and Na-9 group. For example, the Na-7 group is derived from an α amino acid skeleton, but it may be a β amino acid skeleton.

Usually, the method of activation from an amine is not limited to the one containing an SH group as the above-mentioned reaction auxiliary group. Usually, as a method of activation from an amine, it is possible to introduce a hetero atom directly into the amine to improve the reactivity of the amine. For example, hydroxyamine (compound F-1, compound F-4, compound F-5, compound F-7), alkoxyamine (compound F-2, compound F-14, compound F-15, compound F-16), azide (Compound F-3, Compound F-9, Compound F-10, Compound F-11) and the like. As described above, all methods for activating an amino group by introducing a heteroatom that can be a reaction auxiliary group directly or in the vicinity of the amino group with a linker are included in the present application. In the reaction with hydroxyamine, compound F-7, compound F-8, etc. are selected as the active ester moiety. In the reaction with alkoxyamine, compound F-17, compound F-18 and the like are selected. In the reaction with azide, compound F-12, compound F-13 and the like are selected.

R101, R102, R103, R104, R105, R106, and R107 are commonly used amino acid side chains, and are substituents not limited to natural amino acids. That is, a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, and optionally substituted Selected from good aralkyl groups.
More preferably, one of R101 and R102 is a hydrogen atom, one of R103 and R104 is a hydrogen atom, and one of R106 and R107 is a hydrogen atom. The hydrogen atoms are preferably arranged so that they have the same steric configuration as the L-type amino acid.

R105 is selected from an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, and an optionally substituted aralkyl group.

As a combination with an active ester which is a candidate for a crossing unit corresponding to the case where an activated amine as described above is selected, for example, a combination of an amine having a thioester and a thiol in the vicinity, an α ketoester and an azide, and the like can be considered.

Peptide compounds or peptides with various N-terminal introductions of amino acids, amino acid analogs or N-terminal carboxylic acid analogs other than the above methionine by using initiation read-through There is a method that does not require the preparation of a plurality of types of aminoacyl translation initiation tRNAs in the compound library synthesis method. Initiation read through generally means that proteins and peptides are translated from methionine, which is the translation initiation amino acid encoded as an AUC codon. It means a phenomenon in which a translation product from an amino acid encoded by the second and subsequent codons is generated when an attempt is made to start translation from a low unnatural amino acid added to a translation initiation tRNA.

As a method using Initiation 方法 readＮthrough, a triangular unit is encoded at the second codon next to the start codon of mRNA encoding the peptide, and is translated by a translation system that does not contain methionine or translation initiation methionine tRNA. A method for preparing a peptide or peptide library having a terminal trigonal unit can be used. As another report, a method is known in which an N-terminal methionine of a peptide is scraped by the action of an enzyme such as peptide-deformylase and Methionine-aminopeptidase (Non-patent literature: Meinnel, T). ., Et al. Biochimie (1993) 75, 1061-1075, Methionine as translation start signal: A review of the enzymes of the pathway in Escherichia coli.). It is also possible to prepare a library of peptides starting from translation-initiating methionine, and removing N-terminal methionine by allowing Methionine-aminopeptidase to act on this, thereby preparing a library with a random N-terminus. It was also shown that the N-terminal amino acid such as methionine can be removed by cyclization using the second amino acid following translation initiation methionine and subsequent aminopeptidase treatment. As a result, methionine residues are not included in the unit of Scheme A (the number of units is already defined to be determined by the chemical structure in which all post-translational modifications have been completed), and the amino acid residue corresponding to the triangular unit is 2 As a result of the second amino acid encoded, a triangular unit can be encoded by a plurality of codons, so that the degree of freedom is expanded to two or more types and one variable part is added. Since it is possible to use two or more types of amino acids or amino acid analogs as a triangular unit, when preparing the peptide compound or peptide compound library of the present invention, the peptide compound or peptide compound live having increased diversity. A rally can be produced. It is possible to use the same number of amino acids or amino acid analogs as the maximum number of random regions, and to expand to the same number of degrees of freedom as the random regions. Here, the random region means a region where an amino acid or an amino acid analog can be freely selected in the peptide compound of the present invention. Except for this method, a region other than the crossing unit and the triangular unit of Scheme A (that is, a black circle). In this method, triangular units are also random areas. The structural diversity of the black circle unit and the square unit can be secured while maintaining the reactivity with the cross unit of the triangle unit. In other words, using this method, two amino acids (corresponding to the triangle unit and the crossing unit) had to be fixed in the display library for post-translational cyclization, whereas the fixed one It can be reduced to amino acids (crossing units). For example, since most amino acids and amino acid analogs selected in the random region have a main chain amino group, when this is applied to a triangular unit, a random amino acid sequence having an amino group is arranged at the N-terminus. The Among them, amide cyclization can be carried out by selective amide bond formation with a common main chain amino group. For example, it is particularly useful when constructing a display library that does not introduce basic amino acids such as lysine and arginine. From the results of our study, the number of residues having drug likeness is 13 or less, so that the number of units to be fixed decreases from 2 to 1, the number of units in the random region from 11 to 12 Means to increase. The value of increasing the number of residues that can be randomized by one is very high in the sense that it maximizes the degree of freedom under limited conditions to maintain drug likeness.

Specifically, carboxylic acid or carboxylic acid active ester can be translated and introduced into the crossing unit (amino acid intersecting linear part, cyclic part and triangular unit) side chain, so from fixed crossing unit and random amino acid An amide bond can be formed between selected triangular units (Scheme B). When constructing a library, the crossing unit does not have to be one type, and can be selected from two or more types. Specifically, a codon encoding each crossing unit, (amino) acylated tRNA is prepared, and a library can be constructed using mRNA in which the crossing unit codon is arranged at a desired position as a template.
Scheme B

As an example of a method for constructing a library that is cyclized without fixing the N-terminus (triangle unit), an example of amide cyclization can be given (this method can also be used when the N-terminus is fixed). An aspartic acid derivative is taken as an example of the crossing unit, but is not limited thereto, and is selected from, for example, a compound group represented by Compound C-1, Compound C-2, and Compound C-3. Any amino acid or amino acid analog having a carboxylic acid in the side chain, such as an N-alkylated product such as N-methylaspartic acid or a glutamic acid derivative, may be used. (i) It is possible to introduce an amino acid having a carboxylic acid in the side chain and to make it an active ester after translation. For example, aspartic acid itself can be translated and introduced as shown in Compound E-1. The resulting translated peptide can be amide cyclized by a condensation reaction between a carboxylic acid and an N-terminal amine. For example, it can be converted into an active ester such as N-hydroxysuccinimide active ester or HOBt or HOAt as shown in compound E-2. The obtained active ester can be easily reacted with an amine, and as a result, an amide cyclization reaction in which the N-terminal is randomized can be realized. The key to realizing this method is to select a method in which only the carboxylic acid site is active esterified and RNA (such as a nucleic acid site) is not reacted.ア ミ ノ 酸 (ii) An amino acid having a carboxylic acid active ester in the side chain can be introduced by translation, and the active ester can be reacted with an amine. In this method, an active ester such as compound E-2 is preliminarily translated and synthesized. In addition to compound E-2, a benzylthioester such as compound E-3, an arylthioester such as compound E-4, an alkylthioester, and the like can also be introduced by translation. In the case of Compound E-4 and Compound E-3, a phenyl group is taken as an example, but there is no limitation as long as it is an aryl group or a heteroaryl group. In addition, aryl groups and heteroaryl groups can be substituted with, for example, electron-withdrawing groups such as halogen groups, nitro groups, trifluoromethyl groups, and nitrile groups, alkoxy groups such as methoxy groups, and alkyl groups such as methyl groups. Such as an electron donating group. In consideration of the rate of the thioester exchange reaction, the reactivity of the thioester after the exchange reaction with the amine and the selectivity of the side reaction with water, a substituent having a good balance between these is preferable. In addition, examples of the substituent include bulky substituents typified by t-butyl group and isopropyl group, such as a sulfonic acid group and a dimethylamino group that have been used in water and have an affinity for water. Selected from di-substituted amino groups and conversely lipophilic groups such as long-chain alkyl groups in consideration of lipophilicity. Many kinds of substituents such as nitro group, trifluoromethyl group, and halogen may be introduced at the same time. Like compound E-4, a thioaryl active ester activated more than compound E-3 may be introduced in advance. The thioester can be selected from alkyl thioesters in addition to such aralkyl and aryl thioesters. The key to the realization of this method is to have two properties that are stable during translational synthesis and have sufficient reactivity with an amine having no reaction auxiliary group. (iii) Translation of active esters that can ensure sufficient stability during translation synthesis from thioesters, etc., and adding additives after translation to generate more active active esters in the system and react It is also possible to carry out a cyclization reaction with an amine having no auxiliary group. For example, a more active thioester is generated in the system by adding thioester having a more electron deficiency to the thioester introduced by translation from the outside, and cyclized with an amine. For example, in the case of a thioester such as compound E-3, it may be reacted directly with an amino group after translation, but a more reactive thiol such as trifluoromethylphenylthiol may be added to the translation system. Alternatively, the activated ester E-4 may be substituted with a more activated ester and then reacted with an amino group. In addition, for example, various raw materials known to form active esters such as HOBt, HOAt, and HONSu can be added. One type of additive may be selected from these, or two or more types may be selected. An advantage of adding two or more additives is improved reactivity. It is energetically disadvantageous to convert an active ester that is sufficiently stable and translatable in a translation solution into an active ester that is sufficiently reactive with any amino group, and such a reaction may be difficult in one step. . In such a case, once the translatable stable active ester is returned to a more active active ester that can be exchanged, it can be further returned to an active ester sufficiently reactive with any amino group. Become. By using the activation of the active ester in such a multi-stage, an amidation reaction with an amino group having low reactivity may be possible. Substituents may be introduced in these additives such as active esters. For example, substituents such as halogen groups, nitro groups, trifluoromethyl groups, nitrile groups and other electron-withdrawing groups, methoxy groups and the like It may have an electron donating group such as an alkyl group such as an alkoxy group or a methyl group. Bulky substituents typified by t-butyl and isopropyl groups, such as sulfonic acid groups, carboxyl groups, hydroxy groups, and dimethylamino groups in consideration of affinity with water It is selected from a di-substituted amino group, or a highly lipophilic group such as a long-chain alkyl group in consideration of lipophilicity. (iv) A stable active ester is introduced in the same manner as in (iii), a chemical reaction (for example, deprotection reaction) is carried out after translation, and after activation by an intramolecular reaction, an amine having no reaction auxiliary group It is also possible to cause a cyclization reaction. For example, as compound E-5, a more stable thioester is translated and introduced, and after translation, the SS bond is deprotected, and is converted into a more reactive arylthiophenol by intramolecular reaction. May be reacted with an amine. In addition, this problem may be achieved by combining two or more of the concepts (i) to (iv). In this way, as one method for effectively utilizing the Initiation read through method, a more diverse Display library having a druglike cyclization site by reacting an amino group commonly present at the N-terminus with a crossover unit. Can be built.

Among these, (iii) alkylthioesters or benzylthioesters such as alkylthioesters or benzylthioesters, which can ensure sufficient stability during translation synthesis, use active esters that can be translated, and add additives after translation to obtain more active active esters. When it is generated in the system and cyclized with an amine having no reaction auxiliary group, as an active ester used for translational synthesis, for example, aspartic acid side chain carboxylic acid is alkylthioester such as methylthioester (Asp (SMe)) or benzyl Aralkylthioesters such as thioester (Asp (SBn)) can be used.

Examples of additives that are added for the purpose of chemically reacting with a triangular unit having no reaction auxiliary group after translation synthesis as a crossing unit include arylthiols such as 4- (trifluoromethyl) benzenethiol and heteroarylthiols, These may be substituted with an electron-withdrawing group, an electron-donating group, a fat-soluble group, a water-soluble group, or the like, but is preferably an electron-withdrawing group. Examples of the electron-withdrawing group include a trifluoromethyl group, a nitro group, and a fluoro group, and an electron-withdrawing group of about the trifluoromethyl group is preferable.

The addition amount of these thiols is not particularly limited, but is preferably more than 10 mM for the purpose of sufficiently increasing the reactivity, and less than 10 M for the purpose of dissolving the additive. The range of 50 mM to 5 M is more preferable, but 200 mM to 2 M is more preferable. The additive may be added as thiol (acidic), but the acidic part is preferably neutralized by adding an equivalent number of a base such as triethylamine and added as neutral conditions.

Active esters with higher activity may react with various reagents present in the translation reaction system. For example, the amine component (trishydroxymethylaminoethane) in the commonly used Tris buffer can be one of them, so translation synthesis and a buffer for chemical reaction are added in a buffer that does not contain a reactive amine component. It is preferable to do. Examples of such a buffer include HEPES buffer and phosphate buffer.

Also, as the reaction progresses, the side reaction is accompanied by an oxidation reaction in the air (SS formation reaction), avoiding a decrease in the amount of thiol necessary for the progress of the reaction, and the basicity is high and hydrolysis is dominant. In order to avoid this, it is also possible to add a buffer to the reaction translation solution. It is also possible to add a reducing agent such as tris (2-carboxyethyl) phosphine. It is also effective to keep the cyclization reaction away from oxygen in the air as much as possible.

The pH of the solvent during the chemical reaction is preferably 2 to 10 for the purpose of stably presenting RNA. For the purpose of causing the chemical reaction to proceed smoothly, it is preferably 7.8 or more, and for the purpose of suppressing the formation of hydrolysis, it is preferably maintained at 9.2 or less. The reaction conditions may be carried out alone in a reaction translation solution such as PureSystem, and an organic solvent such as DMF or NMP may be added thereto. Moreover, after purifying a translation liquid by column purification etc., you may carry out by changing a solvent. The reaction temperature is not particularly limited as long as it is a range in which a chemical reaction can be usually carried out.

There are no particular limitations on the triangular unit that facilitates the cyclization reaction, and the amino group can be either a primary amine or a secondary amine (for example, an N-alkyl group such as N-methyl). Is not limited. In particular, when the carbon atom next to the amino group is unsubstituted (CH2), either a primary amine or a secondary amine is acceptable. Among the secondary amines, a methyl group is more preferable. When a substituent is present at the carbon atom next to the amino group, a primary amine is more preferable. As the substituent portion, it is more preferable that the β-position is CH2 such as Ala or Phe, such as Val or Thr. In addition, amino acids in which a nitrogen atom and an α-position carbon atom form a 5-membered ring such as proline, and cyclic secondary amines such as a 4-membered ring and a 6-membered ring are also preferable.

In the above, an example of a library construction method in which N-terminal (triangular unit) is cyclized without fixing was described. Amide ring by condensation reaction of amino group and active ester group without using reaction auxiliary group The use of the conversion reaction is not limited to the reaction with the N-terminal main chain amino group. Performed in any combination of the amino group of the side chain of an amino acid or amino acid analog or the amino group of an N-terminal carboxylic acid derivative and the active ester of the side chain of an amino acid or amino acid analog or the active ester of an N-terminal carboxylic acid derivative can do.

Further, in this method, as in Scheme C, a unit having an active ester may be arranged in the triangular unit, an amine side chain may be arranged on the crossing unit side, and the active ester and the amino group may be arranged between the triangular unit and the crossing unit. It may be arranged in either.

Examples of such combinations are described below.
When a unit having an active ester group is selected as the crossing unit, the crossing unit is selected from compound C-1, compound C-2, compound C-3, or compound COH-1, compound COH-2, and compound COH-3. You can also choose. Whenever these six kinds of compounds are selected, it is preferable that the amino acid immediately after the C-terminal side is selected from N-alkylated units. This limitation is for the purpose of avoiding side reactions of aspartimide formation and is a common preferred choice when these six compounds are selected. From the viewpoint of metabolic stability, Compound C-1, or Compound C-2, or Compound C-3 is more preferable. In such a case, the triangular unit can be selected from Compound Na-1, Compound Na-2, and Compound Na-3. When the N-terminal (triangular unit) is not fixed as a triangular unit, a plurality of these compounds can be selected simultaneously.
The side chain amino group may be protected in any of Compound Na-1, Compound Na-2, and Compound Na-3. When protected, the cyclization reaction is carried out after deprotection at the same time or in advance of the cyclization reaction. For the protecting group or deprotection conditions, the method described in this specification can be used.

Ra13 of compound Na-1 can be selected from a hydrogen atom, a C1-C6 alkyl group or an aralkyl group. These may be substituted with a functional group defined by drug-like, such as a hydroxyl group, a fluoro group, and an ether group. Preferably, a hydrogen atom or a methyl group, an ethyl group, an npropyl group, or a benzyl group is preferable.

Compound Ra-2 Ra1 can be selected in the same manner as Ra13. Particularly preferably, it is selected from a hydrogen atom or a methyl group. Ra2 can be selected in the same manner as Ra13, but preferably a hydrogen atom or a methyl group. Particularly preferred is a hydrogen atom. When a hydrogen atom is selected, the steric configuration of the L-type and D-type is allowed as an amino acid. An L-shaped configuration is more preferable. Ra3 can be selected in the same manner as R13. Ra3 and Ra4 may form a ring. Particularly preferred is a hydrogen atom. Ra4 can be selected from a hydrogen atom, an alkyl group, a cycloalkyl group, an aralkyl group, a heteroaryl group, and an aryl group. These may be substituted with a functional group defined by drug-like. Ra4 may form a ring together with Ra1. For example, proline and the like correspond to this ring.

Ra11 of compound Na-3 can be selected in the same manner as Ra13, and particularly preferably a hydrogen atom, or a methyl group, an ethyl group, an npropyl group, or a benzyl group. Particularly preferred is a hydrogen atom. Ra9 can be selected in the same way as Ra4. It is preferably selected from a hydrogen atom, a C1-C6 alkyl group or an aralkyl group. These may be substituted with a functional group defined by drug-like. Further, Ra9 and Ra11 may form a ring. The ring size is preferably a 3- to 8-membered ring. As the ring formation, a 5-membered ring and a 6-membered ring are particularly preferable. As the substituent for Ra9, a hydrogen atom is particularly preferable. Ra10 and Ra12 can be selected in the same manner as Ra4. Preferably, it can select like Ra13. More preferably, either Ra10 or Ra12 is a hydrogen atom. Particularly preferably, both Ra10 and Ra12 are hydrogen atoms. In this case, the N-terminal is preferably selected from an amino acid derivative or an N-terminal carboxylic acid derivative. The presence of an amino group in the N-terminal main chain is disadvantageous in terms of reaction selectivity, and the amino group is retained even after completion of translation modification, resulting in a decrease in drug likeness.

When the crossing unit is selected from Compound C-1, Compound C-2, or Compound C-3, as a triangular unit, if the triangular unit is placed at a position other than the N-terminal and the triangular unit is fixed, the triangular unit is It can be selected from Compound Na-4 or Compound Na-5.

Compound Ra-4 Ra5 can be selected in the same manner as Ra1. Ra6 can be selected in the same manner as Ra2. Ra7 can be selected in the same way as Ra4. More preferred is a hydrogen atom or a methyl group. Particularly preferred is a hydrogen atom. Ra8 is the same as R4, alkylene group of C1-C6 units such as partial structures N-3, N-4, N-5, N-6, N-7, N-8 (not only ortho-substituted but also meta-substituted , Including para substitution). Here, the substituents R13 to R22 can be selected from functional groups that do not react with an active ester or amino group among functional groups selected by drug-like. Preferably, it is selected from a C4 to C6 alkylene unit which may have a substituent, or a partial structure N-6, N-7, N-8 or the like having an aryl group.

<Ra5 of Compound Na-5 can be selected in the same manner as Ra1. Ra6 can be selected in the same manner as Ra2. Ra7 can be selected in the same way as Ra4. More preferred is a hydrogen atom or a methyl group. Particularly preferred is a hydrogen atom. Ra8 is the same as R4, alkylene group of C1-C6 units such as partial structures N-3, N-4, N-5, N-6, N-7, N-8 (not only ortho-substituted but also meta-substituted , Including para substitution). Here, the substituents R13 to R22 can be selected from functional groups that do not react with an active ester or amino group among functional groups selected by drug-like. Preferably, it is selected from a C4 to C6 alkylene unit which may have a substituent, or a partial structure N-6, N-7, N-8 or the like having an aryl group.

The side chain amino group of either Compound Na-4 or Compound Na-5 may be protected. When protected, the cyclization reaction is carried out after deprotection at the same time or in advance of the cyclization reaction. For the protecting group or deprotection conditions, the method described in this specification can be used.

When the crossing unit is selected from Compound C-1, or Compound C-2, and Compound C-3, as a triangular unit, when the triangular unit is arranged at the N-terminus and cyclized with the side chain amino group, The triangular unit can be selected from a wide group of compounds having an amino group and a carboxyl group in addition to the compound Na-4 and the compound Na-5. In translational synthesis, various units can be translationally synthesized as N-terminal carboxylic acid analogs. There is no particular limitation as long as it has an amino group to be amide cyclized and a carboxylic acid to be translated into a peptide. Preferably, the functional group of the divalent unit that joins the amino group and the carboxylic acid group is selected from among drug-like functional groups. An example of such a compound is Compound Na-6. Ra9 of Compound Na-6 may be selected from an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, and an aralkyl group that may be substituted with a drug-like functional group. In addition, -NRCOR 'group (R and R' are drug-like substituents), -OR (R is a drug-like substituent) and NR groups (including R amino acids, dipeptides, tripeptides, etc.) Etc. are allowed.

On the other hand, it is also possible when a unit on the amino group side exists in the crossing unit. For example, when Compound Na-4 or Compound Na-5 is selected, the triangular unit includes Compound C-1, Compound C-2, Compound C-3, Compound COH-1, Compound COH-2, Compound COH-3, etc. Can also be selected. In this case, it is preferable from the viewpoint of drug-like that a straight chain portion is present also on the N-terminal side from the triangular unit, and the N-terminal is selected from N-terminal carboxylic acid derivatives.
For example, when Compound Na-4 or Compound Na-5 is selected, Compound COH-1, Compound COH-2, Compound COH-3, etc. can be selected as the triangular unit. In this case, it is possible that the triangular unit may be present at the N-terminal, or that the straight-chain part is also present on the N-terminal side of the triangular unit and that the N-terminal is selected from N-terminal carboxylic acid derivatives It is preferable from the point of like.

Also, when compound Na-4, compound Na-5, etc. are selected as the intersection unit, compound Ca-1 can also be selected as the triangular unit.

・ As a method for generating straight chain part 2 (branched site), an amino acid analog capable of forming an ester bond by translation synthesis without having an amino group in the main chain, such as α-hydroxycarboxylic acid, and the like Translation and introduction of both amino acids having an amino group side chain which may be protected (the protecting group is not particularly limited as long as it acts as a protecting group for the amino group and gives an amino acid to be translated and synthesized) Post-modification techniques can be used (Scheme E). When applied to a display library, it is selected from the units to be translated. However, peptide compounds obtained by subsequent optimization include those obtained by post-translational modification after translation synthesis even if the peptide compound itself is not translationally synthesized. The side chain of the α-hydroxycarboxylic acid moiety is not particularly limited, but in the method described in Scheme E, Re1 is particularly a hydrogen atom (glycolic acid ( ^HO Gly) itself) or a thiol in the side chain in terms of translational synthesis. Those having a group (SH group) or a protected thiol group are preferred. The configuration of the side chain is not particularly limited, but when a hydrogen atom is present at the α-position, it is more preferable to adopt the same configuration as that of the L-type amino acid. The arrangement of the thiol group or the protected thiol group is not particularly limited, but is arranged at the β-position or γ-position of the OH group (that is, the mercaptomethyl group or mercaptoethyl group where Re1 may be protected). Is particularly preferred. These thiol groups or protected thiol groups are arranged from an optionally substituted alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aralkyl group, aryl group, heteroaryl group of the α-hydroxycarboxylic acid side chain (Added). These α-hydroxycarboxylic acid side chain substituents are more preferably substituents defined on the side chain of a drug-like amino acid except for the added thiol group or protected thiol group.

The type of amino acid having an amino side chain is also an alkylamino group, an alkenylamino group, an alkynylamino group, an aralkylamino group, an arylamino group, a heteroarylamino group, which may be substituted if it has an amino group, Any of the cycloalkylamino groups is possible and is not particularly limited, but may have a thiol group (SH group) or a protected thiol group in the side chain at the same time. These substituents are preferably substituents defined in the side chain of a drug-like amino acid, except for a reaction auxiliary group (for example, SH group). Furthermore, it is more preferable to select from a substituent capable of translational synthesis. One of the hydrogen atoms at the site represented by NH2 in the scheme is specified as a hydrogen atom, and the other hydrogen atom may be substituted with an alkyl group, an alkenyl group, an alkynyl group, an aralkyl group. , An aryl group, a heteroaryl group, and a cycloalkyl group, which may be substituted with a reaction auxiliary group, preferably an NH2 group. These substituents are preferably substituents defined in the side chain of a drug-like amino acid, except for a reaction auxiliary group (for example, SH group). Furthermore, it is more preferable to select from a substituent capable of translational synthesis. a codon encoding an amino acid analog capable of forming an ester bond without an amino group, such as α-hydroxycarboxylic acid (A), an acylated tRNA, a codon encoding an amino acid having an amino group side chain (B), An aminoacylated tRNA is prepared, and mRNA in which a desired number (preferably 0 to 7, more preferably 0 to 2) of random codons is arranged between codon (A) and codon (B). The peptide obtained by translation into a template is first cyclized by the above method or another method. Codon A is arranged N-terminal from codon B. The obtained cyclized peptide (for example, the method of Scheme B or Scheme C can be used) is deprotected as needed when a side chain amino group has a protecting group, and is hydrolyzed or added from the outside. When the ester site is activated by adding an agent, the amide bond is not hydrolyzed, but the ester bond can be hydrolyzed or activated (active esterification, active thioesterification). The peptide having the straight chain part 2 which is a desired branched peptide can be obtained by intramolecular cyclization reaction of the obtained main chain carboxylic acid or active (thio) ester and side chain amine. For example, the additive added from the outside includes a compound group that forms various active esters such as thiol compounds, HONSu, HOBt, and HOAt, or a mixture of two or more of these. In Scheme E, an example was given in which a Re1-substituted hydroxycarboxylic acid was used as codon A and lysine was used as codon B.

When Re1 is a hydrogen atom, in the first cyclization (cyclization of a triangular unit and a crossing unit), the triangular unit may be selected to have a reaction auxiliary group at the amine site. You may choose what you do not have. When a unit having a reaction assisting group is selected as a triangular unit (for example, Compound N-1 or Compound N-2), the first cyclization reaction can easily proceed. In the reaction with a crossover unit having a thioester in the side chain capable of translational synthesis, a reaction additive may or may not be added at a pH of around 7 (translation conditions). On the other hand, when a unit having no reaction auxiliary group is selected as a triangular unit (for example, when Ala or Phe contained in compound Na-2 is selected), in order to proceed the first reaction, trifluoromethylthiophenol It is more preferable to add an additive such as More preferably, the pH is raised to around 7.8 and a reaction time of about 6 to 10 hours is applied at a reaction temperature of about 37 ° C. to 50 ° C. In order to perform the second branching reaction, the amine site may have no reaction auxiliary group, but it is more preferable to have a reaction auxiliary group at the amine site, in which case the NH group must be protected. Is preferred. In this case, it is preferable to add thiol as an ester activator. As the thiol, an alkyl thiol is preferable, and an alkyl thiol having a water-soluble substituent is particularly preferable from the viewpoint of solubility of the additive, and 2-dimethylaminoethane thiol and 2-mercaptoethane sulfonic acid are particularly preferable. The addition amount of these thiols is not particularly limited, but is preferably more than 10 mM for the purpose of sufficiently increasing the reactivity, and less than 10 M for the purpose of dissolving the additive. The range of 100 mM to 5 M is more preferable, but the range of 500 mM to 4 M is more preferable. The selection of the amine moiety is not particularly limited, but the reaction conditions in the case of having deprotection are the same as those described in Scheme F2.

Various methods are possible as variations of the method of Scheme E. For example, using a hydroxycarboxylic acid having an SH group or a protected SH group at the Re1 site, reaction of an SH group or a protected SH group in the vicinity of the amine side chain site or the amine of the protected amine side chain site An amino acid having an auxiliary group introduced therein may be introduced by translation. In this case, when the protecting group imparted to the SH group of the peptide obtained by translation and, if necessary, the amine moiety when protected are deprotected, ester-to-thioester exchange shown in Scheme F easily occurs. . Since the obtained thioester easily reacts with an amine having a reaction assisting group, a peptide having a desired branched peptide (linear portion 2) can be obtained. The SH group of the obtained branched peptide containing an SH group can be easily desulfurized under mild reaction conditions such that RNA does not participate in chemical reactions. Also in this case, an additive may be added from the outside for the purpose of activating the (thio) ester. In the method of Scheme F, the cyclization reaction of the post-translation cyclization part may be performed either before or after the linear chain part 2 formation reaction.
As described above, it may have a reaction auxiliary group such as an SH group or a protected SH group at both the hydroxycarboxylic acid and the amine side chain site, or may have a reaction auxiliary group only on one of them. Or both of them may not have a reaction auxiliary group.

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